Shikonin ameliorates depressive- and anxiogenic-like behaviors in rats via the suppression of inflammation in the hippocampus.

Shikonin is an active naphthoquinone with antioxidative, anti-inflammatory, and anticancer properties. In this study, we investigated the effects of shikonin on depressive- and anxiety-like behaviors in lipopolysaccharide- (LPS-) induced depression and chronic unpredictable mild stress (CUMS) rat models and explored the potential mechanism. First, a 14-day intraperitoneal administration of shikonin (10 mg/kg) significantly decreased immobility time in forced swimming test (FST) and increased open arm entries in elevated plus maze (EPM) test, without affecting line crossings in open field test (OFT), indicating that shikonin has anti-depressant- and anxiolytic-like effects. Second, chronic shikonin administration (10 mg/kg) reversed depressive- and anxiety-like behaviors in LPS-induced and CUMS depression models, as shown in the sucrose preference test (SPT), FST, EPM, and novel object recognition test (NORT). Finally, shikonin significantly reduced the levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) in hippocampus, indicating that the anti-depressant- and anxiolytic-like effects of shikonin are related to the reduction of neuroinflammation in hippocampus. These findings suggest that shikonin exerts anti-depressant- and anxiolytic-like effects via an anti-inflammatory mechanism of shikonin in the hippocampus.

Roles of nucleus accumbens shell small-conductance calcium-activated potassium channels in the conditioned fear freezing.

BACKGROUND: Posttraumatic stress disorder (PTSD), a psychiatric disorder caused by stressful events, is characterized by long-lasting fear memory. The nucleus accumbens shell (NAcS) is a key brain region that regulates fear-associated behavior. Small-conductance calcium-activated potassium channels (SK channels) play a key role in regulating the excitability of NAcS medium spiny neurons (MSNs) but their mechanisms of action in fear freezing are unclear. METHOD: We established an animal model of traumatic memory using conditioned fear freezing paradigm, and investigated the alterations in SK channels of NAc MSNs subsequent to fear conditioning in mice. We then utilized an adeno-associated virus (AAV) transfection system to overexpress the SK3 subunit and explore the function of the NAcS MSNs SK3 channel in conditioned fear freezing. RESULTS: Fear conditioning activated NAcS MSNs with enhanced excitability and reduced the SK channel-mediated medium after-hyperpolarization (mAHP) amplitude. The expression of NAcS SK3 were also reduced time-dependently. The overexpression of NAcS SK3 impaired conditioned fear consolidation without affecting conditioned fear expression, and blocked fear conditioning-induced alterations in NAcS MSNs excitability and mAHP amplitude. Additionally, the amplitudes of mEPSC, AMPAR/NMDAR ratio, and membrane surface GluA1/A2 expression in NAcS MSNs was increased by fear conditioning and returned to normal levels upon SK3 overexpression, indicating that fear conditioning-induced decrease of SK3 expression caused postsynaptic excitation by facilitating AMPAR transmission to the membrane. CONCLUSION: These findings show that the NAcS MSNs SK3 channel plays a critical role in conditioned fear consolidation and that it may influence PTSD pathogenesis, making it a potential therapeutic target against PTSD.

Novel role of AMPK in cocaine reinforcement via regulating CRTC1.

Repeated cocaine exposure causes compensatory neuroadaptations in neurons in the nucleus accumbens (NAc), a region that mediates reinforcing effects of drugs. Previous studies suggested a role for adenosine monophosphate-activated protein kinase (AMPK), a cellular energy sensor, in modulating neuronal morphology and membrane excitability. However, the potential involvement of AMPK in cocaine use disorder is still unclear. The present study employed a cocaine self-administration model in rats to investigate the effect of AMPK and its target cyclic adenosine monophosphate response element binding protein-regulated transcriptional co-activator 1 (CRTC1) on cocaine reinforcement and the motivation for cocaine. We found that intravenous cocaine self-administration significantly decreased AMPK activity in the NAc shell (NAcsh), which persisted for at least 7 days of withdrawal. Cocaine reinforcement, reflected by self-administration behavior, was significantly prevented or enhanced by augmenting or suppressing AMPK activity pharmacologically and genetically, respectively. No difference in sucrose self-administration behavior was found after the same manipulations. The inhibition of AMPK activity in the NAcsh also increased the motivation for cocaine in progressive-ratio schedules of reinforcement, whereas the activation of AMPK had no effect. The knockdown of CRTC1 in the NAcsh significantly impaired cocaine reinforcement, which was rescued by pharmacologically increasing AMPK activity. Altogether, these results indicate that AMPK in the NAcsh is critical for cocaine reinforcement, possibly via the regulation of CRTC1 signaling. These findings may help reveal potential therapeutic targets and have important implications for the treatment of cocaine use disorder and relapse.

Exogenous Carbon Monoxide Produces Rapid Antidepressant- and Anxiolytic-Like Effects.

Carbon monoxide (CO), a byproduct of heme catalyzed by heme oxygenase (HO), has been reported to exert antioxidant and anti-inflammatory actions, and to produce significant neuroprotective effects. The potential effects of CO and even HO on depressive-like behaviors are still poorly understood. Utilizing several approaches including adeno-associated virus (AAV)-mediated overexpression of HO-1, systemic CO-releasing molecules (CO-RMs), CO-rich saline or CO gas treatment procedures in combination with hydrogen peroxide (H2O2)-induced PC12 cell injury model, and lipopolysaccharide (LPS)-induced depression mouse model, the present study aimed to investigate the potential antidepressant- and anxiolytic-like effects of endogenous and exogenous CO administration in vivo and in vitro. The results of in vitro experiments showed that both CO-RM-3 and CO-RM-A1 pretreatment blocked H2O2-induced cellular injuries by increasing cell survival and decreasing cell apoptosis and necrosis. Similar to the effects of CO-RM-3 and CO-RM-A1 pretreatment, AAV-mediated HO-1 overexpression in the dorsal hippocampus produced significant antidepressant-like activities in mice under normal conditions. Further investigation showed that the CO gas treatment significantly blocked LPS-induced depressive- and anxiety-like behaviors in mice. Taken together, our results suggest that the activation of HO-1 and/or exogenous CO administration produces protective effects and exerts antidepressant- and anxiolytic-like effects. These data uncover a novel function of the HO-1/CO system that appears to be a promising therapeutic target for the treatment of depression and anxiety.

Atractylenolide III reduces depressive- and anxiogenic-like behaviors in rat depression models.

Atractylenolide III, a major component of the atractylodes macrocephala Koidz, derived from the rhizoma atractylodes, has been reported to produce various pharmacological effects including anti-aging, anti-inflammation, anti-tumor, and other effects. Growing evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α, are increased in depressed patients. The present study was aimed at investigating the antidepressant- and anxiolytic-like effects of atractylenolide III in lipopolysaccharide (LPS) challenge and chronic unpredictable mild stress (CUMS) rat model. We found that 30 mg/kg of atractylenolide III administered by oral gavage for 14 days, significantly reduced the immobility time in a forced swimming test (FST), but did not alter the number of crossings in an open field test (OFT), respectively. The results indicated that atractylenolide III has an antidepressant-like effect without affecting locomotor activity. We then used the LPS-induced depression model to assess the effects of atractylenolide III on behaviors in FST, sucrose preference test (SPT), and novelty-suppressed feeding test (NSFT). Interestingly, in addition to the antidepressant-like effects, 30 mg/kg of atractylenolide III also produced an anxiolytic-like effect. To further identify the antidepressant- and anxiolytic-like effects of atractylenolide III, we used the CUMS model with 28 consecutive days of the atractylenolide III treatment, followed by the SPT, FST, and NSFT. Atractylenolide III prevented CUMS-induced depressive- and anxiety-like behaviors in rats. To illustrate the underlying possible mechanisms of action of atractylenolide III, we measured the proinflammatory cytokines levels. The results showed that atractylenolide III decreased the proinflammatory cytokines levels in the hippocampus of CUMS exposed rats. In summary, our findings demonstrated that atractylenolide III produces antidepressant- and anxiolytic-like effects in rats, and these effects appear to be mediated by inhibition of hippocampal neuronal inflammation.

BFAR coordinates TGFß signaling to modulate Th9-mediated cancer immunotherapy.

TGFß is essential for the generation of anti-tumor Th9 cells; on the other hand, it causes resistance against anti-tumor immunity. Despite recent progress, the underlying mechanism reconciling the double-edged effect of TGFß signaling in Th9-mediated cancer immunotherapy remains elusive. Here, we find that TGFß-induced down-regulation of bifunctional apoptosis regulator (BFAR) represents the key mechanism preventing the sustained activation of TGFß signaling and thus impairing Th9 inducibility. Mechanistically, BFAR mediates K63-linked ubiquitination of TGFßR1 at K268, which is critical to activate TGFß signaling. Thus, BFAR deficiency or K268R knock-in mutation suppresses TGFßR1 ubiquitination and Th9 differentiation, thereby inhibiting Th9-mediated cancer immunotherapy. More interestingly, BFAR-overexpressed Th9 cells exhibit promising therapeutic efficacy to curtail tumor growth and metastasis and promote the sensitivity of anti-PD-1-mediated checkpoint immunotherapy. Thus, our findings establish BFAR as a key TGFß-regulated gene to fine-tune TGFß signaling that causes Th9 induction insensitivity, and they highlight the translational potential of BFAR in promoting Th9-mediated cancer immunotherapy.

A STING-related prognostic score predicts high-risk patients of colorectal cancer and provides insights into immunotherapy.

BACKGROUND: Targeted therapeutic strategies for advanced colorectal cancer (CRC) have been limited. STING is crucial to the antitumor immunotherapy, for it stimulates IFN signaling to mediate the crosstalk between innate and adaptive immune responses. Emerging evidence suggests that STING also contributes to the prognosis of CRC. However, prognostic models relating to STING have not yet been explored. METHODS: A total of 431 CRC samples from the TCGA database were analyzed to explore the prognostic value of STING-related genes. We trained prognostic models using the multivariate Cox regression. A STING-related prognostic score (SPS) was calculated as the gene expression multiplied by the corresponding coefficients of the final model. A backward stepAIC strategy was adopted to select the optimal model. A nomogram was used to personalize medical decisions for CRC. RESULTS: The expression level of STING was upregulated in the CMS1 subtype (P=0.036). Among STING-related genes, DHX9 (HR =0.72, P=0.01), IRF2 (HR =1.34, P=0.022), and POLR1D (HR =1.23, P=0.038) showed significant prognostic value. The SPS was proven to be an independent risk factor (training: HR =2.9, P=0.00013; validation: HR =3.02, P=0.01), and outperformed random classifiers in identifying high-risk CRC. The high SPS group was characterized by less genomic aberrations, upregulated IL6-JAK-STAT3 and IL2-STAT5 signaling pathways, increased expression of TIM-3, increased infiltration of regulatory T (Treg) cells and T helper 17 (Th17) cells, and decreased infiltration of M0 macrophages. Finally, the nomogram based on the SPS and clinical factors showed good performance in CRC. CONCLUSIONS: SPS is an independent risk factor that could identify high-risk CRC. While ICBs may benefit patients of the CMS1 subtype, for the CMS2, CMS3, and CMS4 subtypes in the high SPS group, STING agonists and immunotherapies targeting the Th17 axis may be beneficial. Finally, the SPS-based nomogram could help advance personalized medical decisions for CRC.

Combined chemotherapy of platinum and fluorouracil promotes T cell-mediated antitumor immunity.

The two-drug combined chemotherapy of platinum and fluorouracil has been reported to efficiently kill tumor cells as the first-line treatment for advanced gastric cancer. However, the effect of these drugs on T cells remains unclear. Here, we showed that T cells including CD4+ T cells and CD8+ T cells of the patients with advanced gastric cancer after platinum and fluorouracil chemotherapy exhibited enhanced ex vivo proliferation ability as compared to that before chemotherapy. In addition, platinum and fluorouracil also promoted the differentiation of human T cells into Th1 and Th9 subtypes and cytotoxic T lymphocytes (CTLs) in vitro and in vivo. Accordingly, the combination therapy greatly suppressed tumor growth with increased tumor infiltration of Th1, Th9, and CTL cells in a mouse tumor model. Moreover, in activated T cells, long-term treatment with these two drugs further facilitates T cell activation along with promoted nuclear factor-κB (NF-κB) activation. Our findings demonstrate a previously unidentified function of platinum and fluorouracil combination chemotherapy in promoting T cell-mediated antitumor immunity.

A pan-cancer analysis of HER2 index revealed transcriptional pattern for precise selection of HER2-targeted therapy.

BACKGROUND: The prevalence of HER2 alterations in pan-cancer indicates a broader range of application of HER2-targeted therapies; however, biomarkers for such therapies are still insufficient and limited to breast cancer and gastric cancer. METHODS: Using multi-omics data from The Cancer Genome Atlas (TCGA), the landscape of HER2 alterations was exhibited across 33 tumor types. A HER2 index was constructed using one-class logistic regression (OCLR). With the predictive value validated in GEO cohorts and pan-cancer cell lines, the index was then applied to evaluate the HER2-enriched expression pattern across TCGA pan-cancer types. FINDINGS: Increased HER2 somatic copy number alterations (SCNAs) could be divided into two patterns, focal- or arm-level. The expression-based HER2 index successfully distinguished the HER2-enriched subtype from the others and provided a stable and superior performance in predicting the response to HER2-targeted therapies both in breast tumor tissue and pan-cancer cell lines. With frequencies varying from 12.0% to 0.9%, tumors including head and neck squamous tumors, gastrointestinal tumors, bladder cancer, lung cancer and uterine tumors exhibited high HER2 indices together with HER2 amplification or overexpression, which may be more suitable for HER2-targeted therapies. The BLCA.3 and HNSC.Basal were the most distinguishable subtypes within bladder cancer and head and neck cancer respectively by HER2 index, implying their potential benefits from HER2-targeted therapies. INTERPRETATION: As a pan-cancer predictive biomarker of HER2-targeted therapies, the HER2 index could help identify potential candidates for such treatment in multiple tumor types by combining with HER2 multi-omics features. The discoveries of our study highlight the importance of incorporating transcriptional pattern into the assessment of HER2 status for better patient selection. FUNDING: The National Key Research and Development Program of China; Clinical Research and Cultivation Project of Shanghai ShenKang Hospital Development Center.

Basolateral amygdala is required for reconsolidation updating of heroin-associated memory after prolonged withdrawal.

Postretrieval extinction procedures are effective nonpharmacological interventions for disrupting drug-associated memories. Nonetheless, the conditioned stimulus (CS) memory retrieval-extinction procedure is ineffective in inhibiting drug craving and relapse after prolonged withdrawal, which significantly undermines its therapeutic potential. In the present study, we showed that, unlike the CS memory retrieval-extinction procedure, noncontingent heroin injections (unconditioned stimulus [UCS]) 1 hour before the extinction sessions decreased the heroin-priming-induced reinstatement, renewal, and spontaneous recovery of heroin seeking after 28 days of withdrawal (ie, remote heroin-associated memories) in rats. The UCS retrieval manipulation induced reactivation of the basolateral amygdala (BLA) after prolonged withdrawal, and this reactivation was absent with the CS retrieval manipulation. Chemogenetic inactivation of the BLA abolished the inhibitory effect of the UCS memory retrieval-extinction procedure on heroin-priming-induced reinstatement after prolonged withdrawal. Furthermore, the combination of chemogenetic reactivation of BLA and CS retrieval-extinction procedure resembled the inhibitory effect of UCS retrieval-extinction procedure on heroin seeking after prolonged withdrawal. We also observed that the inhibitory effect of the UCS retrieval-extinction procedure is mediated by regulation of AMPA receptor endocytosis in the BLA. Our results demonstrate critical engagement of the BLA in reconsolidation updating of heroin-associated memory after prolonged withdrawal, extending our knowledge of the boundary conditions of the reconsolidation of drug-associated memories.

Molecular hydrogen attenuates methamphetamine-induced behavioral sensitization and activation of ERK-ΔFosB signaling in the mouse nucleus accumbens.

Methamphetamine (METH) is one of the most prevalently used illegal psychostimulants in many countries. Continuous exposure to METH leads to behavioral sensitization in animals, which can be used as a behavioral model with many mechanisms in common with relapse in humans. Molecular hydrogen has recently gained attention for its potential as a novel healthcare product with preventive and therapeutic applicability to a wide range of pathological conditions. However, it remains unclear whether and, if so, how hydrogen regulates METH-induced behavioral abnormalities. In the present study, we investigated the roles of molecular hydrogen on the acquisition and transfer of METH-induced behavioral sensitization and the accompanying changes in ERK phosphorylation and ΔFosB activation in the nucleus accumbens (NAc) of mice. To this end, male C57BL/6 mice received METH (0.1, 0.5 and 1.0 mg/kg, i.p.) injections for 7 days followed by a METH challenge (0.1, 0.5 and 1.0 mg/kg, i.p.) after a 7-day transfer period. Molecular hydrogen, delivered through a hydrogen-rich saline (HRS) injection (10 mL/kg, i.p., 3-h interval), was administered during the acquisition and transfer periods. We found that HRS administration was able to inhibit the acquisition and transfer of 0.1 and 0.5 mg/kg METH-induced behavioral sensitization to a certain extent, thereby attenuating the expression of behavioral sensitization. The HRS injections alone did not induce any obvious changes in locomotor activity in mice. Intriguingly, the increases in pERK and ΔFosB in the NAc, which accompanied the METH-induced behavioral sensitization, were also attenuated by the HRS treatments. Due to the anti-oxidative function of molecular hydrogen, the HRS injections reduced METH-induced reactive oxygen species and malondialdehyde generation in the NAc. These results suggest that molecular hydrogen serves as an anti-oxidative agent with potentially therapeutic applicability to the treatment of METH addicts.

Calpain-GRIP Signaling in Nucleus Accumbens Core Mediates the Reconsolidation of Drug Reward Memory.

Exposure to drug-paired cues causes drug memories to be in a destabilized state and interfering with memory reconsolidation can inhibit relapse. Calpain, a calcium-dependent neutral cysteine protease, is involved in synaptic plasticity and the formation of long-term fear memory. However, the role of calpain in the reconsolidation of drug reward memory is still unknown. In the present study, using a conditioned place preference (CPP) model, we found that exposure to drug-paired contextual stimuli induced the activation of calpain and decreased the expression of glutamate receptor interacting protein 1 (GRIP1) in the nucleus accumbens (NAc) core, but not shell, of male rats. Infusions of calpain inhibitors in the NAc core immediately after retrieval disrupted the reconsolidation of cocaine/morphine cue memory and blocked retrieval-induced calpain activation and GRIP1 degradation. The suppressive effect of calpain inhibitors on the expression of drug-induced CPP lasted for at least 14 d. The inhibition of calpain without retrieval 6 h after retrieval or after exposure to an unpaired context had no effects on the expression of reward memory. Calpain inhibition after retrieval also decreased cocaine seeking in a self-administration model and this effect did not recover spontaneously after 28 d. Moreover, the knock-down of GRIP1 expression in the NAc core by lentivirus-mediated short-hairpin RNA blocked disruption of the reconsolidation of drug cue memories that was induced by calpain inhibitor treatment. These results suggest that calpain activity in the NAc core is crucial for the reconsolidation of drug reward memory via the regulation of GRIP1 expression.SIGNIFICANCE STATEMENT Calpain plays an important role in synaptic plasticity and long-term memory consolidation, however, its role in the reconsolidation of drug cue memory remains unknown. Using conditioned place preference and self-administration procedures, we found that exposure to drug-paired cues induced the activation of calpain and decreased glutamate receptor interacting protein 1 (GRIP1) expression in the nucleus accumbens (NAc) core. The inhibition of calpain activity in the NAc core immediately after retrieval disrupted the reconsolidation of cocaine/morphine cue memory that was blocked by prior GRIP1 knock-down. Our findings indicate that calpain-GRIP signaling is essential for the restabilization process that is associated with drug cue memory and the inhibition of calpain activity may be a novel strategy for the prevention of drug relapse.

Molecular hydrogen increases resilience to stress in mice.

The inability to successfully adapt to stress produces pathological changes that can lead to depression. Molecular hydrogen has anti-oxidative and anti-inflammatory activities and neuroprotective effects. However, the potential role of molecular hydrogen in stress-related disorders is still poorly understood. The present study aims to investigate the effects of hydrogen gas on resilience to stress in mice. The results showed that repeated inhalation of hydrogen-oxygen mixed gas [67%:33% (V/V)] significantly decreased both the acute and chronic stress-induced depressive- and anxiety-like behaviors of mice, assessed by tail suspension test (TST), forced swimming test (FST), novelty suppressed feeding (NSF) test, and open field test (OFT). ELISA analyses showed that inhalation of hydrogen-oxygen mixed gas blocked CMS-induced increase in the serum levels of corticosterone, adrenocorticotropic hormone, interleukin-6, and tumor necrosis factor-α in mice exposed to chronic mild stress. Finally, inhalation of hydrogen gas in adolescence significantly increased the resilience to acute stress in early adulthood, which illustrates the long-lasting effects of hydrogen on stress resilience in mice. This was likely mediated by inhibiting the hypothalamic-pituitary-adrenal axis and inflammatory responses to stress. These results warrant further exploration for developing molecular hydrogen as a novel strategy to prevent the occurrence of stress-related disorders.

Selective Inhibition of Amygdala Neuronal Ensembles Encoding Nicotine-Associated Memories Inhibits Nicotine Preference and Relapse.

BACKGROUND: Nicotine craving and relapse often occurs after reactivation of nicotine reward memories. We recently developed a memory retrieval-reconsolidation interference procedure in which reactivating nicotine reward memories by acute exposure to nicotine (the unconditioned stimulus [UCS]) and then pharmacologically interfering with memory reconsolidation decreased relapse to nicotine seeking in rats and nicotine craving in smokers. Here, we investigated underlying mechanisms. METHODS: In the first series of experiments, we trained rats for nicotine-induced conditioned place preference (CPP) or nicotine self-administration and ventricularly microinjected them with the protein synthesis inhibitor anisomycin immediately after UCS-induced memory retrieval. In the second series of experiments, we used tyramide-amplified immunohistochemistry-fluorescence in situ hybridization to examine neural ensembles in the basolateral amygdala (BLA) reactivated by nicotine conditioned stimulus- or UCS-induced memory retrieval. We then used the Daun02 chemogenetic inactivation procedure to selectively inhibit the nicotine UCS-reactivated BLA neuronal ensembles. RESULTS: Ventricular injections of the anisomycin immediately after nicotine UCS memory retrieval inhibited subsequent nicotine CPP and relapse to operant nicotine seeking after short or prolonged abstinence. More important, within BLA, distinct neuronal ensembles encoded pavlovian CPP and operant self-administration reward memories and nicotine (the UCS) injections in the home cage reactivated both neuronal ensembles. Daun02 chemogenetic inactivation of the nicotine-reactivated ensembles inhibited both nicotine CPP and relapse to nicotine seeking. CONCLUSIONS: Results demonstrate that the nicotine UCS-induced memory retrieval manipulation reactivates multiple nicotine reward memories that are encoded by distinct BLA neuronal ensembles that play a role in nicotine preference and relapse.

AMPK signaling in the nucleus accumbens core mediates cue-induced reinstatement of cocaine seeking.

Relapse to drug seeking can be caused by exposure to drug-associated cues, provoking drug craving even after prolonged abstinence. Recent studies demonstrated that AMP-activated protein kinase (AMPK) regulates neuronal morphology and membrane excitability in neurons. Here, we investigated the role of AMPK activity in the nucleus accumbens (NAc) in relapse to cocaine seeking. We found that exposure to drug-related cues reinstated cocaine-seeking behavior and increased AMPK and p70s6k phosphorylation in the NAc core but not shell. Augmenting AMPK activity by intra-NAc core infusions of the AMPK activator 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) or adenovirus expressing constitutively active subunits of AMPK decreased cue-induced reinstatement of cocaine seeking and inhibited the mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways. In contrast, inhibition of AMPK activity by intra-NAc core infusions of the AMPK inhibitor compound C or adenovirus expressing dominant-negative subunits of AMPK increased cue-induced reinstatement of cocaine seeking and enhanced mTORC1 and ERK1/2 activity. The regulation of AMPK activity in the NAc shell had no effect on cue-induced cocaine seeking. Altogether, these results indicate that AMPK activity in the NAc core is critical for the cue-induced reinstatement of cocaine seeking, which may be mediated by mTORC1 and ERK1/2 signaling.

Effect of Selective Inhibition of Reactivated Nicotine-Associated Memories With Propranolol on Nicotine Craving.

Importance: A relapse into nicotine addiction during abstinence often occurs after the reactivation of nicotine reward memories, either by acute exposure to nicotine (a smoking episode) or by smoking-associated conditioned stimuli (CS). Preclinical studies suggest that drug reward memories can undergo memory reconsolidation after being reactivated, during which they can be weakened or erased by pharmacological or behavioral manipulations. However, translational clinical studies using CS-induced memory retrieval-reconsolidation procedures to decrease drug craving reported inconsistent results. Objective: To develop and test an unconditioned stimulus (UCS)-induced retrieval-reconsolidation procedure to decrease nicotine craving among people who smoke. Design, Setting, and Participants: A translational rat study and human study in an academic outpatient medical center among 96 male smokers (aged 18- 45 years) to determine the association of propranolol administration within the time window of memory reconsolidation (after retrieval of the nicotine-associated memories by nicotine UCS exposure) with relapse to nicotine-conditioned place preference (CPP) and operant nicotine seeking in rats, and measures of preference to nicotine-associated CS and nicotine craving among people who smoke. Intervention: The study rats were injected noncontingently with the UCS (nicotine 0.15 mg/kg, subcutaneous) in their home cage, and the human study participants administered a dose of propranolol (40 mg, per os; Zhongnuo Pharma). Main Outcomes and Measures: Nicotine CPP and operant nicotine seeking in rats, and preference and craving ratings for newly learned and preexisting real-life nicotine-associated CS among people who smoke. Results: Sixty-nine male smokers completed the experiment and were included for statistical analysis: 24 in the group that received placebo plus 1 hour plus UCS, 23 who received propranolol plus 1 hour plus UCS, and 22 who received UCS plus 6 hours plus propranolol. In rat relapse models, propranolol injections administered immediately after nicotine UCS-induced memory retrieval inhibited subsequent nicotine CPP and operant nicotine seeking after short (CPP, d = 1.72, 95% CI, 0.63-2.77; operant seeking, d = 1.61, 95% CI, 0.59-2.60) or prolonged abstinence (CPP, d = 1.46, 95% CI, 0.42-2.47; operant seeking: d = 1.69, 95% CI, 0.66-2.69), as well as nicotine priming-induced reinstatement of nicotine CPP (d = 1.28, 95% CI, 0.27-2.26) and operant nicotine seeking (d = 1.61, 95% CI, 0.59-2.60) after extinction. Among the smokers, oral propranolol administered prior to nicotine UCS-induced memory retrieval decreased subsequent nicotine preference induced by newly learned nicotine CS (CS1, Cohen d = 0.61, 95% CI, 0.02-1.19 and CS2, d = 0.69, 95% CI, 0.10-1.28, respectively), preexisting nicotine CS (d = 0.57, 95% CI, -0.02 to 1.15), and nicotine priming (CS1, d = 0.82, 95% CI, 0.22-1.41 and CS2, d = 0.78, 95% CI, 0.18-1.37, respectively; preexisting nicotine CS, d = 0.92, 95% CI, 0.31-1.52), as well as nicotine craving induced by the preexisting nicotine CS (d = 0.64, 95% CI, 0.05-1.22), and nicotine priming (d = 1.15, 95% CI, 0.52-1.76). Conclusions and Relevance: In rat-to-human translational study, a novel UCS-induced memory retrieval-reconsolidation interference procedure inhibited nicotine craving induced by exposure to diverse nicotine-associated CS and nicotine itself. This procedure should be studied further in clinical trials.

AMPK Signaling in the Dorsal Hippocampus Negatively Regulates Contextual Fear Memory Formation.

Both the formation of long-term memory (LTM) and dendritic spine growth that serves as a physical basis for the long-term storage of information require de novo protein synthesis. Memory formation also critically depends on transcription. Adenosine monophosphate-activated protein kinase (AMPK) is a transcriptional regulator that has emerged as a major energy sensor that maintains cellular energy homeostasis. However, still unknown is its role in memory formation. In the present study, we found that AMPK is primarily expressed in neurons in the hippocampus, and then we demonstrated a time-dependent decrease in AMPK activity and increase in mammalian target of rapamycin complex 1 (mTORC1) activity after contextual fear conditioning in the CA1 but not CA3 area of the dorsal hippocampus. Using pharmacological methods and adenovirus gene transfer to bidirectionally regulate AMPK activity, we found that increasing AMPK activity in the CA1 impaired the formation of long-term fear memory, and decreasing AMPK activity enhanced fear memory formation. These findings were associated with changes in the phosphorylation of AMPK and p70s6 kinase (p70s6k) and expression of BDNF and membrane GluR1 and GluR2 in the CA1. Furthermore, the prior administration of an mTORC1 inhibitor blocked the enhancing effect of AMPK inhibition on fear memory formation, suggesting that this negative regulation of contextual fear memory by AMPK in the CA1 depends on the mTORC1 signaling pathway. Finally, we found that AMPK activity regulated hippocampal spine growth associated with memory formation. In summary, our results indicate that AMPK is a key negative regulator of plasticity and fear memory formation.

Neuropeptide Trefoil Factor 3 Reverses Depressive-Like Behaviors by Activation of BDNF-ERK-CREB Signaling in Olfactory Bulbectomized Rats.

The trefoil factors (TFFs) are a family of three polypeptides, among which TFF1 and TFF3 are widely distributed in the central nervous system. Our previous study indicated that TFF3 was a potential rapid-onset antidepressant as it reversed the depressive-like behaviors induced by acute or chronic mild stress. In order to further identify the antidepressant-like effect of TFF3, we applied an olfactory bulbectomy (OB), a classic animal model of depression, in the present study. To elucidate the mechanism underlying the antidepressant-like activity of TFF3, we tested the role of brain-derived neurotrophic factor (BDNF)-extracellular signal-related kinase (ERK)-cyclic adenosine monophosphate response element binding protein (CREB) signaling in the hippocampus in the process. Chronic systemic administration of TFF3 (0.1 mg/kg, i.p.) for seven days not only produced a significant antidepressant-like efficacy in the OB paradigm, but also restored the expression of BDNF, pERK, and pCREB in the hippocampal CA3. Inhibition of BDNF or extracellular signal-related kinase (ERK) signaling in CA3 blocked the antidepressant-like activity of TFF3 in OB rats. Our findings further confirmed the therapeutic effect of TFF3 against depression and suggested that the normalization of the BDNF-ERK-CREB pathway was involved in the behavioral response of TFF3 for the treatment of depression.

Monocyte mediated brain targeting delivery of macromolecular drug for the therapy of depression.

Leukocytes can cross intact blood-brain barrier under healthy conditions and in many neurological diseases, including psychiatric diseases. In present study, a cyclic RGD (cRGD) peptide with high affinity for integrin receptors of leukocytes was used to modify liposomes. The cRGD-modified liposomes (cRGDL) showed high affinity for monocytes in vitro and in vivo and co-migrated across in vitro BBB model with THP-1. The trefoil factor 3 (TFF3), a macromolecular drug, was rapidly and persistently delivered to brain for at least 12 h when loaded into cRGDL while 2.8-fold increase in drug concentration in basolateral amygdala regions related to depression was observed. A systemic administration of cRGDL-TFF3 mimicked antidepressant-like effect of direct intra-basolateral amygdala administration of TFF3 solution in rats subjected to chronic mild stress. The effective dual-brain targeting delivery resulting from the combination and co-migration of cRGDL with leukocyte cross BBB may be a promising strategy for targeted brain delivery. FROM THE CLINICAL EDITOR: In an effort to treat depression, brain targeted delivery via monocyte-cRGD liposome complexes capable of crossing the intact BBB was performed in this study in a murine model. Similar approaches may be helpful in the treatment of other neuropsychiatric conditions.

The mammalian target of rapamycin pathway in the basolateral amygdala is critical for nicotine-induced behavioural sensitization.

Repeated exposure to nicotine increases psychomotor activity. Long-lasting neural plasticity changes that contribute to the nicotine-induced development of locomotor sensitization have been identified. The mammalian target of rapamycin complex 1 (mTORC1) signalling pathway is involved in regulating the neuroplasticity of the central nervous system. In this study, we examined the role of mTORC1 in the amygdala in nicotine-induced locomotor sensitization. Rapamycin, an inhibitor of mTORC1, was infused into the basolateral amygdala (BLA) and central amygdala (CeA) or systemically administered to investigate the role of the mTORC1 in the development and expression of nicotine-induced locomotor sensitization. We found that locomotor activity progressively increased during the initiation of nicotine-induced locomotor sensitization and the expression of nicotine sensitization was induced by nicotine challenge injection (0.35 mg/kg s.c.) after five days of withdrawal. The initiation of nicotine-induced locomotor sensitization was accompanied by the increased phosphorylated level of mTORC1 downstream target proteins including p-p70s6k and p-4EBP in the BLA, but not CeA. Intra-BLA infusion or systemic administration of rapamycin blocked locomotor activity. Increased p-p70s6k and p-4EBP were also observed in the expression of nicotine sensitization, which was demonstrated to be inhibited by systemic rapamycin administration. Our findings indicated that mTORC1 activity in the BLA, but not the CeA, mediated the initiation and expression of nicotine-induced locomotor sensitization, and may become a potential target for the treatment of nicotine addiction.