Ameliorating effects of Acanthopanax koreanum extract and components on nicotine dependence and withdrawal symptoms.

Tobacco smoking is a serious health problem in society. While smoking rates are declining, smoking remains a serious risk to national health. Currently, there are several medications available to aid in smoking cessation. However, these medications have the disadvantages of low success rates in smoking cessation and various side effects. Therefore, natural-based smoking cessation aids are being suggested as a good alternative due to their accessibility and minimal side effects. The roots and stems of Acanthopanax koreanum (AK) Nakai, a plant that is native to Jeju Island, South Korea, have traditionally been used as tonic and sedatives. Moreover, eleutheroside B and chlorogenic acid are the main components of AK stem extract. In the present study, we investigated the effect of 70% ethanol AK extract and its components on ameliorating nicotine dependence and withdrawal symptoms by using behavioural tests in mice. In addition, alterations in the dopaminergic and DRD1-EPAC-ERK-CREB pathways were observed using dopamine ELISA and western blotting using mouse brains. Our findings demonstrate that the AK extract and its components effectively mitigated the effects of nicotine treatment in behavioural tests. Furthermore, it normalized the dopamine concentration and the expression level of nicotine acetylcholine receptor α7. Additionally, it was observed that AK extract and its components led to the normalization of DRD1, ERK and CREB expression levels. These results indicate that AK extract exhibits effects in ameliorating nicotine dependence behaviour and alleviating withdrawal symptoms. Moreover, EB and CGA are considered potential marker components of AK extract.

Extracts of Prunella vulgaris Enhanced Pentobarbital-Induced Sleeping Behavior in Mice Potentially via Adenosine A2A Receptor Activity.

The increasing prevalence of sleep dysregulation cases has prompted the search for effective and safe sleep-enhancing agents. Numerous medications used in the treatment of sleep disorders function by enhancing γ-aminobutyric acid neurotransmitter activity. Unfortunately, these substances may induce significant adverse effects in chronic users, such as dependence and motor behavior impairments. Consequently, there is a growing interest in exploring therapeutic sleep-enhancing agents derived from natural sources, with the anticipation of causing less severe side effects. Prunella vulgaris (PV), a perennial plant indigenous to South Korea, exhibits various pharmacological effects, likely attributed to its chemical composition. Rosmarinic acid, one of its components, has previously demonstrated sleep-potentiating properties, suggesting the potential for PV to exhibit similar pharmacological effects. This study aims to investigate the potential effects of repeated administration of PV extract on the sleep behavior, brainwave activity, sleep-wake cycle, and physiological behavior of mice. Findings indicate that PV extracts exhibit sleep-enhancing effects in mice, characterized by prolonged sleep duration and a reduced onset time of pentobarbital-induced sleep. However, PV extracts only reduced alpha wave powers, with minor alterations in wakefulness and rapid-eye-movement sleep duration. In contrast to diazepam, PV extracts lack adverse effects on locomotor activity, motor coordination, or anxiety in mice. Receptor-binding assay and caffeine treatment support the potential involvement of adenosine A2A receptors in the effects of PV, suggesting distinct mechanisms of action compared to diazepam, despite both exhibiting sleep-altering effects. Overall, our results suggest that PV holds promise as a potential source of sleep-aiding agents.

The dopaminergic alterations induced by 4-F-PCP and 4-Keto-PCP may enhance their drug-induced rewarding and reinforcing effects: Implications for abuse.

Novel psychoactive substances remain the popular recreational drugs of use over the years. They continue to bypass government restrictions due to their synthesis and modifications. Recent additions to the lists are the 4-F-PCP and 4-Keto-PCP, analogs of the drug phencyclidine (PCP) known to induce adverse effects and abuse potential. However, studies on the abuse potential of 4-F-PCP and 4-Keto-PCP remain scarce. The rewarding and reinforcing effects of the drugs were assessed using conditioned place preference (CPP), self-administration, and locomotor sensitization tests. Dopamine (DA) receptor antagonists (SCH23390 and haloperidol) were administered during CPP to evaluate the involvement of the mesolimbic dopaminergic system. DA-related protein expression in the nucleus accumbens (NAcc) and ventral tegmental area (VTA) was measured. Additionally, phosphorylated cyclic-adenosine monophosphate-activated protein (AMP) response element-binding (p-CREB) protein, deltaFosB (∆FosB), and brain-derived neurotrophic factor (BDNF) protein levels in the NAcc were measured to assess the addiction neural plasticity effect of the drugs. Both 4-F-PCP and 4-Keto-PCP-induced CPP and self-administration; however, only 4-F-PCP elicited locomotor sensitization. Treatment with DA receptor antagonists (SH23390 and haloperidol) inhibited the 4-F- and 4-Keto-induced CPP. Both substances altered the levels of DA receptor D1 (DRD1), thyroxine hydroxylase (TH), DA receptor D2 (DRD2), p-CREB, ∆FosB, and BDNF. The results suggest that 4-F-PCP and 4-Keto-PCP may induce abuse potential in rodents via alterations in dopaminergic system accompanied by addiction neural plasticity.

Estrogen receptor/androgen receptor transcriptional activation of benzophenone derivatives and integrated approaches to testing and assessment (IATA) for estrogenic effects.

Estrogen receptor (ER) and androgen receptor (AR) transactivation assays for the benzophenone compounds (BPs) were performed using hERα-HeLa-9903 cells for ER and MMTV/22Rv1_GR-KO cells for AR. Results showed that some BPs, such as BP-1, BP-2, 4OH-BP, 4DHB, and 4-MBP, showed agonistic activity on ER with a higher RPCmax than 1 nM 17-ß estradiol. The other BPs (BP, BP-3, BP-6, BP-7, and BP-8) showed low RPCmax in accordance with the OECD Test guideline (TG) 455 criteria, with BP-4 as the only ER-negative. However, the potency of the BPs was at least 1000 times less than the reference chemical, 17-ß-estradiol. None of the BPs exhibited agonistic activity on AR except BP-2 which showed a small increase in activity. For further evaluation of the estrogenic effect of BPs based on the integrated approaches to testing and assessment (IATA) approach, existing data on ER binding, steroidogenesis, MCF-7 cell proliferation, and in vivo uterotrophic assays were collected and evaluated. There seemed to be a close association between the in vitro data on BPs, especially ER transcriptional activity, and the in vivo results of increased uterine weight. This case study implied that integrated approaches using in vitro data can be a useful tool for the prediction of in vivo data for estrogenic effects, without the need for additional animal toxicity tests.

Integrated Assessment for the Estrogenic Effects of Pyrethroid Compounds: Defining the Molecular Initiating Events and Key Events for the Adverse Outcome Pathway.

Pyrethroids, which are derived from natural insecticides found in chrysanthemum flowers, are widely utilized in various sectors, including agriculture, forestry, horticulture, and personal insect protection. Due to their widespread use, concerns have arisen regarding their potential estrogenic effects on female reproductive health. This review aims to address data gaps and inconsistencies in previous studies by defining molecular initiating events and key events within the adverse outcome pathway associated with pyrethroid-induced estrogenic effects. To achieve this, we propose utilizing Integrated Approaches to Testing and Assessment (IATA), which incorporate in vitro assays and in vivo assessments to comprehensively investigate the estrogenic effects of pyrethroids. An initial search was conducted in the PubMed database to identify relevant articles. Subsequently, the findings were classified according to the IATA strategy. This review provides an overview of the current understanding of pyrethroids and their estrogenic effects, identifies data gaps, and highlights the use of IATA in existing studies on the estrogenic effects of various pyrethroids. It emphasizes the urgent need for comprehensive research on the estrogenic effects of pyrethroids and highlights the importance of standardized testing methods like IATA to accurately assess their impact on human and environmental health. By promoting the use of Integrated Testing Strategies (ITSs) and addressing data gaps, researchers and regulators can enhance the accuracy of assessments, ensuring better protection of human and environmental health from the potential estrogenic effects of pyrethroid exposure.

Heukharang (Lactuca sativa L.) extracts enhanced the sleep behavior of mice: potential involvement of adenosine A1 and A2A receptors.

A significant proportion of the world's population suffers from insomnia, a disorder characterized by complications in initiating and maintaining sleep. Many medications used to treat insomnia target the γ-aminobutyric acid (GABA) neurotransmitter system. However, these substances, such as benzodiazepines, induce significant adverse consequences, including dependence and memory impairment, after prolonged use. Thus, current studies are aimed at developing therapeutic hypnotics derived from natural sources that may cause less severe side effects. Heukharang is a variety of lettuce from Korea that was discovered to contain sleep-promoting compounds. Therefore, we investigated the potential effects of sub-chronic administration of Heukharang extract (FSD-LS) on sleep behavior (pentobarbital-induced sleeping test), brain wave activity and sleep architecture (electroencephalography), and physiological behavior (open-field test and rota-rod) in mice, along with radioligand binding assays (GABAA, adenosine A1 and A2A receptors). We found that FSD-LS prolonged the total sleep duration and reduced the onset time of sleep, and enhanced delta wave power and non-rapid eye movement (NREM) sleep duration, all indicating persistent sleep-enhancing effects. FSD-LS lacked adverse effects on the spontaneous locomotor activity and motor coordination of mice, unlike diazepam. Pharmacological blocking using caffeine and bicuculline supported the possible involvement of adenosine receptors in the sleep-promoting effects of FSD-LS, with partial contribution from GABA receptor activity. Overall, our study recommends FSD-LS as a potential source for the development of sleep-aiding therapeutics.

Impact and Interrelationships of Striatal Proteins, EPHB2, OPRM1, and PER2 on Mild Cognitive Impairment.

With the global increase in life expectancy, there has been a rise in the incidence of cognitive impairments attributed to diverse etiologies. Notably, approximately 50% of individuals diagnosed with mild cognitive impairment (MCI) progress to dementia within 3 years. However, the precise mechanisms underlying MCI remain elusive. Therefore, this study aimed to elucidate potential mechanisms implicated in MCI utilizing Per2 knockout (KO) mice, which have previously been shown to have cognitive deficits. Behavioral (Y-maze, Barnes maze) and molecular (electrophysiology, RNA sequencing, western blot, and immunofluorescence) experiments were conducted in Per2 KO and wild-type (WT) mice. Per2 KO mice exhibited impaired spatial working memory in the Y-maze and Barnes maze. However, there were no significant group differences in hippocampal long-term potentiation (LTP) between Per2 KO and WT mice, whereas striatal LTP in Per2 KO mice was lower compared to WT mice. In RNA sequencing analysis, 58 genes were downregulated and 64 genes were upregulated in the striatum of Per2 KO mice compared to WT mice. Among the differentially expressed genes, four genes (Chrm2, EphB2, Htr1b, Oprm1) were identified. Optimal expression levels of EPHB2 and OPRM1 were found to significantly enhance cognitive performance in mice. Additionally, Per2 KO mice exhibited reduced EPHB2-NMDAR-LTP and OPRM-mTOR signaling, along with elevated amyloid beta (Aß) levels, when compared to WT mice. However, these alterations were reversed upon administration of morphine treatment. Striatal OPRM1-mTOR signaling, EPHB2-NMDAR-LTP signaling, and Aß expression levels may exert a combined effect on MCI under the control of Per2 expression.

Korean Red Ginseng extract attenuates alcohol-induced addictive responses and cognitive impairments by alleviating neuroinflammation.

Background: Alcohol is one of the most commonly used psychoactive drugs. Due to its addictive characteristics, many people struggle with the side effects of alcohol. Korean Red Ginseng (KRG) is a traditional herbal medicine that is widely used to treat various health problems. However, the effects and mechanisms of KRG in alcohol-induced responses remain unclear. Therefore, the purpose of this study was to investigate the effects of KRG in alcohol-induced responses. Methods: We investigated two aspects: alcohol-induced addictive responses and spatial working memory impairments. To determine the effects of KRG in alcohol-induced addictive responses, we performed conditioned place preference tests and withdrawal symptom observations. To assess the effects of KRG in alcohol-induced spatial working memory impairment, Y-maze, Barnes maze, and novel object recognition tests were performed using mice after repeated alcohol and KRG exposure. To investigate the potential mechanism of KRG activity, gas chromatography-mass spectrometry and western blot analysis were performed. Results: KRG-treated mice showed dose-dependent restoration of impaired spatial working memory following repeated alcohol exposure. Furthermore, withdrawal symptoms to alcohol were reduced in mice treated with KRG and alcohol. The PKA-CREB signaling pathway was activated after alcohol administration, which was reduced by KRG. However, the levels of inflammatory cytokines were increased by alcohol and decreased by KRG. Conclusion: Taken together, KRG may alleviate alcohol-induced spatial working memory impairments and addictive responses through anti-neuroinflammatory activity rather than through the PKA-CREB signaling pathway.

The differential vulnerabilities of Per2 knockout mice to the addictive properties of methamphetamine and cocaine.

With the pervasive occurrence of substance abuse worldwide, unraveling the neuropharmacology of drugs of abuse, such as psychostimulants, is undeniably essential. Mice lacking Period 2 (Per2), a gene associated with the biological time-regulating system or circadian rhythm, have been proposed as a potential animal model for drug abuse vulnerability, demonstrating a greater preference for methamphetamine (METH) reward than wild-type (WT) mice. However, the responses of Per2 knockout (KO) mice to the reinforcing effects of METH or other psychostimulants are yet to be established. In this study, the responses of WT and Per2 KO mice to various psychostimulants via intravenous self-administration were determined, along with their behaviors in METH- or cocaine (COC)-induced conditioned place preference and spontaneous locomotion in the open-field test. Per2 KO mice exhibited greater addiction-like responses to METH and 5-EAPB (1-(1-benzofuran-5-yl)-N-ethylpropan-2-amine), but their responses to COC and dimethocaine were comparable to WT mice, indicating a divergent influence of Per2 deficiency on abuse susceptibility to specific psychostimulants. To potentially define the underlying mechanism for this phenotype, 19 differentially expressed genes were identified, through RNA sequencing, which might respond specifically to repeated METH, but not COC, administration in the mouse striatum and were narrowed down to those previously associated with immediate early genes or synaptic plasticity. The correlation between locomotor activity and mRNA expression levels revealed a moderate correlation between METH-induced behavior and Arc or Junb expression in Per2 KO mice only, suggesting their essential role that may lead to the higher vulnerability of Per2 KO mice to METH, but not COC. These findings indicate a potentially unique effect of Per2 expression level on the involvement of Arc and Junb in determining specific vulnerabilities to drugs, and possibly including abuse potential.

Serotonin 2C receptors are also important in head-twitch responses in male mice.

RATIONALE: Serotonergic psychedelics exert their effects via their high affinity for serotonin (5-HT) receptors, particularly through activating 5-HT2A receptors (5-HT2AR), employing the frontal cortex-dependent head-twitch response (HTR). Although universally believed to be so, studies have not yet fully ascertained whether 5-HT2AR activation is the sole initiator of these psychedelic effects. This is because not all 5-HT2AR agonists exhibit similar pharmacologic properties. OBJECTIVE: This study aims to identify and discriminate the roles of 5-HT2AR and 5-HT2CR in the HTR induced by Methallylescaline (MAL) and 4-Methyl-2,5,ß-trimethoxyphenethylamine (BOD) in male mice. Also, an analysis of their potential neurotoxic properties was evaluated. METHODS: Male mice treated with MAL and BOD were evaluated in different behavioral paradigms targeting HTR and neurotoxicity effects. Drug affinity, pharmacological blocking, and molecular analysis were also conducted to support the behavioral findings. The HTR induced by DOI has been extensively characterized in male mice, making it a good positive control for this study, specifically for comparing the pharmacological effects of our test compounds. RESULTS: The activation of 5-HT2CR, alone or in concert with 5-HT2AR, produces a comparable degree of HTRs (at a dose of 1 mg·kg-1), with divergent 5-HT2CR- and 5-HT2AR-Gqα11-mediated signaling and enhanced neurotoxic properties (at a dose of 30 mg·kg-1) coupled with activated pro-inflammatory cytokines. These findings show these compounds' potential psychedelic and neurotoxic effects in male mice. CONCLUSION: These findings showed that while 5-HT2AR is the main initiator of HTR, the 5-HT2CR also has a distinct property that renders it effective in inducing HTR in male mice.

4-F-PCP, a Novel PCP Analog Ameliorates the Depressive-Like Behavior of Chronic Social Defeat Stress Mice via NMDA Receptor Antagonism.

Major depressive disorder is a leading cause of disability in more than 280 million people worldwide. Monoamine-based antidepressants are currently used to treat depression, but delays in treatment effects and lack of responses are major reasons for the need to develop faster and more efficient antidepressants. Studies show that ketamine (KET), a PCP analog, produces antidepressant effects within a few hours of administration that lasts up to a week. However, the use of KET has raised concerns about side effects, as well as the risk of abuse. 4 -F-PCP analog is a novel PCP analog that is also an NMDA receptor antagonist, structurally similar to KET, and might potentially elicit similar antidepressant effects, however, there has been no study on this subject yet. Herein, we investigate whether 4-F-PCP displays antidepressant effects and explored their potential therapeutic mechanisms. 4-F-PCP at 3 and 10 mg/kg doses showed antidepressant-like effects and repeated treatments maintained its effects. Furthermore, treatment with 4-F-PCP rescued the decreased expression of proteins most likely involved in depression and synaptic plasticity. Changes in the excitatory amino acid transporters (EAAT2, EAAT3, EAAT4) were also seen following drug treatment. Lastly, we assessed the possible side effects of 4-F-PCP after long-term treatment (up to 21 days). Results show that 4-F-PCP at 3 mg/kg dose did not alter the cognitive function of mice. Overall, current findings provide significant implications for future research not only with PCP analogs but also on the next generation of different types of antidepressants.

Hippocampal dentate gyri proteomics reveals Wnt signaling involvement in the behavioral impairment in the THRSP-overexpressing ADHD mouse model.

Children with attention-deficit/hyperactivity disorder (ADHD) often struggle with impaired executive function, temporal processing, and visuospatial memory, hallmarks of the predominantly inattentive presentation (ADHD-PI), subserved by the hippocampus. However, the specific genes/proteins involved and how they shape hippocampal structures to influence ADHD behavior remain poorly understood. As an exploratory tool, hippocampal dentate gyri tissues from thyroid hormone-responsive protein overexpressing (THRSP OE) mice with defining characteristics of ADHD-PI were utilized in proteomics. Integrated proteomics and network analysis revealed an altered protein network involved in Wnt signaling. Compared with THRSP knockout (KO) mice, THRSP OE mice showed impaired attention and memory, accompanied by dysregulated Wnt signaling affecting hippocampal dentate gyrus cell proliferation and expression of markers for neural stem cell (NSC) activity. Also, combined exposure to an enriched environment and treadmill exercise could improve behavioral deficits in THRSP OE mice and Wnt signaling and NSC activity. These findings show new markers specific to the ADHD-PI presentation, converging with the ancient and evolutionary Wnt signaling pathways crucial for cell fate determination, migration, polarity, and neural patterning during neurodevelopment. These findings from THRSP OE mice support the role of Wnt signaling in neurological disorders, particularly ADHD-PI presentation.

Deletion of Cryab increases the vulnerability of mice to the addiction-like effects of the cannabinoid JWH-018 via upregulation of striatal NF-κB expression.

Synthetic cannabinoids have exhibited unpredictable abuse liabilities, especially self-administration (SA) responses in normal rodent models, despite seemingly inducing addiction-like effects in humans. Thus, an efficient pre-clinical model must be developed to determine cannabinoid abuse potential in animals and describe the mechanism that may mediate cannabinoid sensitivity. The Cryab knockout (KO) mice were recently discovered to be potentially sensitive to the addictive effects of psychoactive drugs. Herein, we examined the responses of Cryab KO mice to JWH-018 using SA, conditioned place preference, and electroencephalography. Additionally, the effects of repeated JWH-018 exposure on endocannabinoid- and dopamine-related genes in various addiction-associated brain regions were examined, along with protein expressions involving neuroinflammation and synaptic plasticity. Cryab KO mice exhibited greater cannabinoid-induced SA responses and place preference, along with divergent gamma wave alterations, compared to wild-type (WT) mice, implying their higher sensitivity to cannabinoids. Endocannabinoid- or dopamine-related mRNA expressions and accumbal dopamine concentrations after repeated JWH-018 exposure were not significantly different between the WT and Cryab KO mice. Further analyses revealed that repeated JWH-018 administration led to possibly greater neuroinflammation in Cryab KO mice, which may arise from upregulated NF-κB, accompanied by higher expressions of synaptic plasticity markers, which might have contributed to the development of cannabinoid addiction-related behavior in Cryab KO mice. These findings signify that increased neuroinflammation via NF-κB may mediate the enhanced addiction-like responses of Cryab KO mice to cannabinoids. Altogether, Cryab KO mice may be a potential model for cannabinoid abuse susceptibility.

Regulation of clock and clock-controlled genes during morphine reward and reinforcement: Involvement of the period 2 circadian clock.

BACKGROUND: Morphine abuse is a devastating disorder that affects millions of people worldwide, and literature evidence indicates a relationship between opioid abuse and the circadian clock. AIM: We explored morphine reward and reinforcement using mouse models with Per2 gene modifications (knockout (KO); overexpression (OE)). METHODS: Mice were exposed to various behavioral, electroencephalographic, pharmacological, and molecular tests to assess the effects of morphine and identify the underlying mechanisms with a focus on reward and reinforcement and the corresponding involvement of circadian and clock-controlled gene regulation. RESULTS: Per2 deletion enhances morphine-induced analgesia, locomotor sensitization, conditioned place preference (CPP), and self-administration (SA) in mice, whereas its overexpression attenuated these effects. In addition, reduced withdrawal was observed in Per2 KO mice, whereas an augmented withdrawal response was observed in Per2 OE mice. Moreover, naloxone and SCH 23390 blocked morphine CPP in Per2 KO and wild-type (WT) mice. The rewarding (CPP) and reinforcing effects (SA) observed in morphine-conditioned and morphine self-administered Per2 KO and WT mice were accompanied by activated µ-opioid and dopamine D1 receptors and TH in the mesolimbic (VTA/NAcc) system. Furthermore, genetic modifications of Per2 in mice innately altered some clock genes in response to morphine. CONCLUSION: These findings improve our understanding of the role of Per2 in morphine-induced psychoactive effects. Our data and those obtained in previous studies indicate that targeting Per2 may have applicability in the treatment of substance abuse.

Per2 Expression Regulates the Spatial Working Memory of Mice through DRD1-PKA-CREB Signaling.

Several individuals worldwide show cognitive impairment due to various reasons, including a prolonged lifespan and an altered lifestyle. Various causes, such as broken circadian rhythms and dopamine-related factors, have been proposed to be involved in the development of cognitive impairment. However, the underlying pathways remain elusive. Humans with circadian misalignment often face cognitive impairments, and animals with mutations in circadian rhythm-related genes display impaired cognitive functions. To analyze this in detail, this study aimed to investigate the pathways potentially involved in cognitive impairment using Period2 (Per2) transgenic animals. Spatial working memory performance in Per2 knockout (KO) and wild-type mice was assessed using the Barnes maze and Y-maze. The dopamine-related protein expression levels in the hippocampus were measured by Western blotting and enzyme-linked immunosorbent assay (ELISA). Per2 KO mice exhibited impaired spatial working memory, and the expression levels of dopamine receptor D1 (DRD1), protein kinase A (PKA), and cAMP response element-binding protein (CREB) were higher in Per2 KO mice than in control mice. Additionally, DRD1 expression levels were inversely proportional to those of PER2. Thus, memory tests were again conducted after administration of the DRD1 antagonist SCH-23390. Per2 KO mice recovered from memory impairment, and the levels of PKA and CREB decreased after treatment. The effects of Aß on memory in Per2 mice were also investigated, and we found the increased Aß levels did not influence the memory performance of Per2 mice after SCH-23390 treatment. These results indicate that Per2 expression levels might influence spatial working memory performance via DRD1-PKA-CREB-dependent signaling.

Differentially Expressed Genes in Period 2-Overexpressing Mice Striatum May Underlie Their Lower Sensitivity to Methamphetamine Addiction-Like Behavior.

Previous reports have demonstrated that genetic mechanisms greatly mediate responses to drugs of abuse, including methamphetamine (METH). The circadian gene Period 2 (Per2) has been previously associated with differential responses towards METH in mice. While the behavioral consequences of eliminating Per2 have been illustrated previously, Per2 overexpression has not yet been comprehensively described; although, Per2-overexpressing (Per2 OE) mice previously showed reduced sensitivity towards METH-induced addiction-like behaviors. To further elucidate this distinct behavior of Per2 OE mice to METH, we identified possible candidate biomarkers by determining striatal differentially expressed genes (DEGs) in both drug-naïve and METH-treated Per2 OE mice relative to wild-type (WT), through RNA sequencing. Of the several DEGs in drug naïve Per2 OE mice, we identified six genes that were altered after repeated METH treatment in WT mice, but not in Per2 OE mice. These results, validated by quantitative real-time polymerase chain reaction, could suggest that the identified DEGs might underlie the previously reported weaker METH-induced responses of Per2 OE mice compared to WT. Gene network analysis also revealed that Asic3, Hba-a1, and Rnf17 are possibly associated with Per2 through physical interactions and predicted correlations, and might potentially participate in addiction. Inhibiting the functional protein of Asic3 prior to METH administration resulted in the partial reduction of METH-induced conditioned place preference in WT mice, supporting a possible involvement of Asic3 in METH-induced reward. Although encouraging further investigations, our findings suggest that these DEGs, including Asic3, may play significant roles in the lower sensitivity of Per2 OE mice to METH.

Senescence and impaired DNA damage responses in alpha-synucleinopathy models.

α-Synuclein is a crucial element in the pathogenesis of Parkinson's disease (PD) and related neurological diseases. Although numerous studies have presented potential mechanisms underlying its pathogenesis, the understanding of α-synuclein-mediated neurodegeneration remains far from complete. Here, we show that overexpression of α-synuclein leads to impaired DNA repair and cellular senescence. Transcriptome analysis showed that α-synuclein overexpression led to cellular senescence with activation of the p53 pathway and DNA damage responses (DDRs). Chromatin immunoprecipitation analyses using p53 and γH2AX, chromosomal markers of DNA damage, revealed that these proteins bind to promoters and regulate the expression of DDR and cellular senescence genes. Cellular marker analyses confirmed cellular senescence and the accumulation of DNA double-strand breaks. The non-homologous end joining (NHEJ) DNA repair pathway was activated in α-synuclein-overexpressing cells. However, the expression of MRE11, a key component of the DSB repair system, was reduced, suggesting that the repair pathway induction was incomplete. Neuropathological examination of α-synuclein transgenic mice showed increased levels of phospho-α-synuclein and DNA double-strand breaks, as well as markers of cellular senescence, at an early, presymptomatic stage. These results suggest that the accumulation of DNA double-strand breaks (DSBs) and cellular senescence are intermediaries of α-synuclein-induced pathogenesis in PD.

Alpha-Synuclein Inclusion Formation in Human Oligodendrocytes.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by presence of α-synuclein-positive inclusions in the cytoplasm of oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are considered an integral part of the pathogenesis of MSA, leading to demyelination and neuronal demise. What is most puzzling in the research fields of GCIs is the origin of α-synuclein aggregates in GCIs, since adult oligodendrocytes do not express high levels of α-synuclein. The most recent leading hypothesis is that GCIs form via transfer and accumulation of α-synuclein from neurons to oligodendrocytes. However, studies regarding this subject are limited due to the absence of proper human cell models, to demonstrate the entry and accumulation of neuronal α-synuclein in human oligodendrocytes. Here, we generated mature human oligodendrocytes that can take up neuronderived α-synuclein and form GCI-like inclusions. Mature human oligodendrocytes are derived from neural stem cells via "oligosphere" formation and then into oligodendrocytes, treating the cells with the proper differentiation factors at each step. In the final cell preparations, oligodendrocytes consist of the majority population, while some astrocytes and unidentified stem cell-like cells were present as well. When these cells were exposed to α-synuclein proteins secreted from neuron-like human neuroblastoma cells, oligodendrocytes developed perinuclear inclusion bodies with α-synuclein immunoreactivity, resembling GCIs, while the stem cell-like cells showed α-synuclein-positive, scattered puncta in the cytoplasm. In conclusion, we have established a human oligodendrocyte model for the study of GCI formation, and the characterization and use of this model might pave the way for understanding the pathogenesis of MSA.

1-Phenylcyclohexan-1-amine hydrochloride (PCA HCl) alters mesolimbic dopamine system accompanied by neuroplastic changes: A neuropsychopharmacological evaluation in rodents.

The recreational use of N-methyl-D-aspartate (NMDA) antagonist phencyclidine (PCP) and ketamine have grown rapidly due to their psychotomimetic properties. These compounds induce both non-fatal and fatal adverse effects and despite the enhanced regulation, they are continuously synthesized and are being sold in the illegal drug market, including 1-phenylcyclohexan-1-amine hydrochloride (PCA). Therefore, we evaluated its abuse potential through the conditioned-place preference (CPP), self-administration, and locomotor sensitization paradigms. Pretreatment with SCH 2 3390 and haloperidol was also performed during a CPP test. We used ELISA to measure dopamine (DA) levels and western blotting to determine effects on the DA-related proteins as well as on phosphorylated CREB, deltaFosB, and brain-derived neurotrophic factor (BDNF) in the ventral tegmental area (VTA) and nucleus accumbens (NAc). Finally, we examined the effects on brain wave activity using electroencephalography (EEG). PCA induced CPP in mice and was self-administered by rats, suggesting that PCA has rewarding and reinforcing properties. PCA increased locomotor of mice on the first treatment and challenge days. SCH 23390 and haloperidol blocked the CPP. PCA altered the DA, tyrosine hydroxylase, dopamine D1 and D2 receptors as well as p-CREB and deltaFosB. Also, PCA altered the delta and gamma waves in the brain, which were then normalized by SCH 2 3390 and haloperidol. The present findings indicate that PCA may induce abuse potential through the dopaminergic system and probably accompanied with alterations in brain wave activity which is similar to that of other psychotomimetic NMDA antagonists. We advocate thorough monitoring of PCP analogs as they pose potential harm to public health.

Gene Expression Profiling in the Striatum of Per2 KO Mice Exhibiting More Vulnerable Responses against Methamphetamine.

Drug addiction influences most communities directly or indirectly. Increasing studies have reported the relationship between circadian-related genes and drug addiction. Per2 disrupted mice exhibited more vulnerable behavioral responses against some drugs including methamphetamine (METH). However, its roles and mechanisms are still not clear. Transcriptional profiling analysis in Per2 knockout (KO) mice may provide a valuable tool to identify potential genetic involvement and pathways in enhanced behavioral responses against drugs. To explore the potential genetic involvement, we examined common differentially expressed genes (DEGs) in the striatum of drug naïve Per2 KO/wild-type (WT) mice, and before/after METH treatment in Per2 KO mice, but not in WT mice. We selected 9 common DEGs (Ncald, Cpa6, Pklr, Ttc29, Cbr2, Egr2, Prg4, Lcn2, and Camsap2) based on literature research. Among the common DEGs, Ncald, Cpa6, Pklr, and Ttc29 showed higher expression levels in drug naïve Per2 KO mice than in WT mice, while they were downregulated in Per2 KO mice after METH treatment. In contrast, Cbr2, Egr2, Prg4, Lcn2, and Camsap2 exhibited lower expression levels in drug naïve Per2 KO mice than in WT mice, while they were upregulated after METH treatment in Per2 KO mice. qRT-PCR analyses validated the expression patterns of 9 target genes before/after METH treatment in Per2 KO and WT mice. Although further research is required to deeply understand the relationship and roles of the 9 target genes in drug addiction, the findings from the present study indicate that the target genes might play important roles in drug addiction.