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This study investigated the butyrylcholinesterase (BChE) inhibitory activity of harmane (1), naucledine (2), and dihydrodeglycocadambine (3) isolated from fractions F7 and F9 of Ochreinauclea maingayi. Both fractions demonstrated significant inhibition, exceeding 80%, against BChE at 100 µg/mL. Compound 2, is the most potent inhibitor, exhibiting an IC50 value of 22.08 µM, followed by 1 and 3 (IC50 23.96 and 30.32 µM, respectively). Docking studies revealed that 1 and 2 effectively bind to BChE, with binding energies of -51.24 and -57.17 kcal/mol, respectively. Kinetic analysis of 2 indicated mixed-mode inhibition of BChE, with a Ki of 6.08 µM. In the paralysis assay, 1 showed a weak delay in paralysis and reduced the paralysis ratio from 72.59 ± 4.7% to 60.00 ± 7.0% (12.59% reduction) followed by 2 with 70.00 ± 1.7% (2.59% reduction) compared with negative standard (DMSO 0.1%) on human amyloid ß-protein in a transgenic Caenorhabditis elegans (CL4176) model.

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Infectious disease caused by methicillin-resistant Staphylococcus aureus (MRSA) seriously threatens public health. The design of antimicrobial peptide mimics (AMPMs) based on natural products (NPs) is a new strategy to kill MRSA and slow the development of drug resistance recently. Here, we reported the design and synthesis of novel AMPMs based on harmane skeleton. Notably, compound 9b exhibited comparable or even better anti-MRSA activity in vitro and in vivo with minimum inhibitory concentration (MIC) of 0.5-2 µg/mL than the positive drug vancomycin. The highly active compound 9b not only showed low cytotoxicity, no obvious hemolysis and good plasma stability, but also presented low tendency of developing resistance. Anti-MRSA mechanism revealed that compound 9b could destroy cell wall structure by interacting with lipoteichoic acid and peptidoglycan, cause membrane damage by depolarization, increased permeability and destructed integrity, reduce cell metabolic activity by binding to lactate dehydrogenase (LDH), interfere cellular redox homeostasis, and bind to DNA. Overall, compound 9b killed the MRSA by multi-target mechanism, which provide a promising light for combating the growing MRSA resistance.

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The ß-carboline motif is common in drug discovery and among numerous biologically active natural products. However, its synthetic preparation relies on multistep sequences and heavily depends on the type of substitution required in the core of the desired ß-carboline target. Herein, we demonstrate that this structural motif can be accessed with the microwave-assisted electrocyclic cyclization of heterotrienic aci (alkylideneazinic acid) forms of 3-nitrovinylindoles. The reaction can start with 3-nitrovinylindoles themselves under two sets of conditions. The first one involves microwave irradiation of butanolic solutions of 3-nitrovinylindoles, whereas the second one consists of prior Boc protection of indolic nitrogen, where the protecting group cleanly comes off during the course of the reaction. Alternatively, the reaction can start with 3-nitrovinylindoles prepared in situ using various processes. Finally, the reaction may utilize indoles with ß-nitrostyrenes, likely involving the intermediacy of spirocyclic oxazolines, which rearrange to similar heterotrienic systems undergoing cyclization to ß-carbolines. As part of this study, several natural products, namely, alkaloids norharmane, harmane, and eudistomin N, were synthesized.

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Altered lipid patterns in Caenorhabditis elegans (C. elegans) resulting from exposure to harmane remain to be explored. In this study, untargeted lipidomics was carried out to elucidate the effects of acute exposure to harmane on the lipidome of C. elegans. Exposure to the compound was evaluated based on the reproduction ability of the worms at 0.1 and 1 µg/mL. No significant effects of harmane were observed at these concentrations. Furthermore, we found that the modulatory effects of harmane on the lipidome of C. elegans at 1 µg/mL were lipid class dependent. In particular, harmane-treated worms were enriched in triglycerides and fatty acids, regardless of the degree of saturation. Glycerophospholipids were generally down-regulated. Furthermore, functional analyses suggested that there was a reduction in lipid membrane bilayer-related terms, and in some related to the mitochondria, and endoplasmic reticulum of C. elegans when treated with harmane. Lipid droplets and storage appeared to be up-regulated. In conclusion, our findings suggest that harmane exposure affects the lipidome of C. elegans in a sophisticated manner. Further investigations are required to elucidate the molecular mechanisms underlying these lipid pattern changes.

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Fusarium oxysporum is a widely distributed soil-borne pathogenic fungus that can cause medicinal herbs and crops to wither or die, resulting in great losses and threat to public health. Due to the emergence of drug-resistance and the decline of the efficacy of antifungal pesticides, there is an urgent need for safe, environmentally friendly, and effective fungicides to control this fungus. Plant-derived natural products are such potential pesticides. Extracts from seeds of Peganum harmala have shown antifungal effects on F. oxysporum but their antifungal mechanism is unclear. In vitro antifungal experiments showed that the total alkaloids extract and all five ß-carboline alkaloids (ßCs), harmine, harmaline, harmane, harmalol, and harmol, from P. harmala seeds inhibited the growth of F. oxysporum. Among these ßCs, harmane had the best antifungal activity with IC50 of 0.050 mg/mL and MIC of 40 µg/mL. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results revealed that the mycelia and spores of F. oxysporum were morphologically deformed and the integrity of cell membranes was disrupted after exposure to harmane. In addition, fluorescence microscopy results suggested that harmane induced the accumulation of ROS and increased the cell death rate. Transcriptomic analysis showed that the most differentially expressed genes (DEGs) of F. oxysporum treated with harmane were enriched in catalytic activity, integral component of membrane, intrinsic component of membrane, and peroxisome, indicating that harmane inhibits F. oxysporum growth possibly through damaging cell membrane and ROS accumulation via regulating steroid biosynthesis and the peroxisome pathway. The findings provide useful insights into the molecular mechanisms of ßCs of P. harmala seeds against F. oxysporum and a reference for understanding the application of ßCs against F. oxysporum in medicinal herbs and crops.

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Heat treatment of meat can lead to the formation of carcinogenic organic compounds. The influence of dried fruits on the formation of non-polar heterocyclic aromatic amines (carbolines) and nitrogen derivatives of polycyclic aromatic hydrocarbons (azaarenes) in roasted pork loin was elucidated. Two hundred grams of fruit per 1 kg of meat were used as stuffing. Carbolines, derivatives of pyridoimidazole and pyridoindole, and azaarenes (benzoacridines and dibenzoacridines) were determined by means of high-performance liquid chromatography with fluorescence detection. The total concentration of six δ-, γ- and α-carbolines in roasted pork loin was 1.3 ng/g. This content decreased by 64%, 58%, and 54% in pork loin stuffed with prunes, apricots, and cranberries, respectively. Concentrations of ß-carbolines (harmane and norharmane) increased under the influence of added fruits. The norharmane content increased the most, from 2.2 ng/g in the control sample to 12.3 ng/g in meat prepared with cranberries. The harmane content increased from 1.0 ng/g to 3.6 ng/g in meat with prunes. The total concentration of azaarenes (two benzoacridines and dibenzo[a,c]acridine), which was close to 0.1 ng/g, decreased in dishes with prunes and apricots by 54% and 12%, respectively. Azaarenes were not found in samples of meat stuffed with cranberries.

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Nicotine is the primary addictive component in cigarette smoke, and dopamine release induced by nicotine is considered a significant cause of persistent smoking and nicotine dependence. However, the effects of nicotine replacement therapy on smoking cessation were less effective than expected, suggesting that other non-nicotine constituents may potentiate the reinforcing effects of nicotine. Harmane is a potent, selective monoamine oxidase A (MAO-A) inhibitor found in cigarette smoke, but showed no effect on nicotine self-administration in previous studies, possibly due to the surprisingly high doses used. In the present study, we found that harmane potentiated nicotine self-administration on the fixed ration schedule at the dose related to human cigarette smoking by the synergistic effects in up-regulating genes in addiction-related pathways, and the effect was reduced at doses 10 times higher or lower than the smoking-related dose. The smoking-related dose of harmane also enhanced the increase of locomotor activity induced by nicotine, accompanied by increased dopamine basal level and dopamine release in the nucleus accumbens through MAO-A inhibition. Our findings provided new evidence for the important role of non-nicotine ingredients of tobacco products in smoking addiction.

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Insights into binding efficacy and thermodynamic aspects of small molecules are important for rational drug designing and development. Here, the interaction of Harmane (Har), a very important bioactive indole alkaloid, with AT and GC hairpin duplex-DNAs has been reported using various biophysical tools. Detailed molecular mechanism with special emphasis on binding nature, base specificity, and thermodynamics have been elucidated via probing nucleic acids with varying base compositions. Har bound to both the DNA strands exhibited hypochromic effect in absorbance whereas bathochromic and hypochromic effects in fluorescence spectra. The binding constants estimated were in the order of 105 M-1 (higher for GC sequence compared with AT) with 1:1 stoichiometry. Noncooperative binding mode has been observed via intercalation in both the cases. The thermodynamic profile was obtained from temperature-dependent fluorescence experiments. Both Har-AT and Har-GC complexations were exothermic in nature associated with positive entropy and negative enthalpy changes. Salt-dependent studies revealed that the binding interaction was governed by nonpolyelectrolytic and hydrophobic interaction forces. The ligand-induced structural perturbation of the DNA structures was evident from the circular dichroism data. Molecular modelling data indicated towards the involvement of hydrophobic forces and hydrogen bonding.

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DNA is one of the major molecular targets for a broad range of anticancer drugs. Hence, interaction studies involving cellular DNA and small molecules can be highly beneficial as they often lead to rational and efficient drug design. In this study, the binding interaction of Harmane (a naturally occurring, bioactive indole alkaloid) with two natural polymeric DNAs, that is, Calf thymus (CT) DNA and Herring testis (HT) DNA has been elucidated using biophysical techniques. A ground state, 1:1 complexation, was revealed by steady-state fluorescence spectroscopy. The thermodynamic profile and energetics of the associated reaction were evaluated by temperature-dependent fluorescence spectroscopy. The spontaneity of the binding was confirmed by the negative ΔG° values in both cases. Negative enthalpy change, along with stronger positive entropic contribution, indicated the dominant electrostatic nature of the interaction and finally the entropy-driven exothermic binding process throughout. Salt-dependent studies further demonstrated the significant contribution of electrostatic interactions in ligand binding toward DNA. Infrared data substantiated the structural information of the said interactions, leading to the exploration of the structure-function relationship.

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Thermal treatment of protein-rich food can lead to the formation of biologically active heterocyclic aromatic amines (HAAs). One of the methods to learn how to reduce the content as well as the influence of these compounds on heath is the study of factors inhibiting their synthesis. In the current investigation, the effect of onion and garlic on the formation of six possibly carcinogenic non-polar HAAs (α-, γ- and δ-carbolines) and two co-mutagenic ß-carbolines (harmane and norharmane) was evaluated by comparing their contents in meat and gravy samples obtained from pan-fried pork dishes prepared in the presence and absence of these vegetables. Carbolines were isolated from food samples by solid phase extraction. The quantitative analysis was performed by high-performance liquid chromatography with fluorescence detection. The concentrations of individual compounds in dishes prepared without added vegetables ranged from 0.02 ng g-1 (3-amino-1,4-dimethyl-5 H-pyrido(4,3-b)indole; Trp-P-1) to 10.1 ng g-1 of meat (2-amino-9 H-pyrido[2,3-b]indole; AαC). Onion (30 g/100 g of meat) and garlic (15 g/100 g of meat) lowered the total content (in meat and gravy) of the α-, δ- and γ-carbolines in the range from 52% to 87%. In contrast, onion caused an increase in the norharmane concentration both in meat and gravy. The percentage of carbolines in the gravies (assuming that their total content in meat and gravy is 100%) was higher in dishes prepared with onion and garlic than in dishes without these seasonings.

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Monoamine oxidases (MAO) are a valuable class of mitochondrial enzymes with a critical role in neuromodulation. In this study, we investigated the effect of natural MAO inhibitors on novel environment-induced anxiety by using the zebrafish novel tank test (NTT). Because zebrafish spend more time at the bottom of the tank when they are anxious, anxiolytic compounds increase the time zebrafish spend at the top of the tank and vice versa. Using this paradigm, we found that harmane, norharmane, and 1,2,3,4-tetrahydroisoquinoline (TIQ) induce anxiolytic-like effects in zebrafish, causing them to spend more time at the top of the test tank and less time at the bottom. 2,3,6-trimethyl-1,4-naphtoquinone (TMN) induced an interesting mix of both anxiolytic- and anxiogenic-like effects during the first and second halves of the test, respectively. TIQ was unique in having no observable effect on general movement. Similarly, a reference MAO inhibitor clorgyline-but not pargyline-increased the time spent at the top in a concentration-dependent manner. We also demonstrated that the brain bioavailability of these compounds are high based on the ex vivo bioavailability assay and in silico prediction models, which support the notion that the observed effects on anxiety-like behavior in zebrafish were most likely due to the direct effect of these compounds in the brain. This study is the first investigation to demonstrate the anxiolytic-like effects of MAO inhibitors on novel environment-induced anxiety in zebrafish.

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Rats were fed a high-fat diet and treated with acrylamide (21 mg/kg BW), harmane (7 mg/kg BW), NƐ-(carboxymethyl)lysine (CML; 10 mg/kg BW), or the co-accumulation of the three chemicals to investigate the effects of harmful Maillard reaction products (MRPs) generated by heat processing of potatoes. Damage to the liver, kidney, gastrocnemius, and nervous system was confirmed by serum biochemical testing and histopathology examination after six weeks of feeding. Serum metabolomic analysis was performed using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). The metabolites detected by GC-TOF-MS were processed using chemometric techniques, and the results were evaluated using a t-test (p < 0.05) and calculated variable importance in projection (VIP) value (>1.5). Significant changes (increase or decrease) were identified in 13 metabolites, compared with the control. These metabolites were gluconolactone, tyrosol, 5-hydroxytryptophan, erythrose 4-phosphate, quinolinic acid, 2-ketobutyric acid, tyrosine, 3-hydroxy-3-methylglutaric acid, desaminotyrosine, L-cysteine, ribonic acid, cysteamine, and tryptophan. Among these, cysteamine and tryptophan are metabolites unique to the co-accumulation of all three test compounds in the treated rats. This study demonstrates that these harmful MRPs exert toxic effects on the liver, kidney, and nervous system via different metabolic pathways. Moreover, co-accumulation of these harmful MRPs caused differing toxic effects and changes in a variety of metabolites. These changes may contribute to the interaction of those harmful MRPs and also affect toxicity at the metabolic level.

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Background: Elevated tissue levels of the tremor-producing neurotoxin, harmane, have been detected in patients with essential tremor (ET) in the USA and Spain. Recently, a study in the Faroe Islands similarly noted an elevation in blood harmane concentrations in probable and definite ET cases. The underlying mechanism is not understood. Possible mechanisms include increased dietary consumption (esp. through cooked meats), impaired metabolism, or increased endogenous production of harmane. To investigate this issue further, we conducted a population-based study in the Faroe Islands to examine meat consumption and meat cooking practices in ET cases and controls. Methods: 1,328 Faroese adults were screened for tremor and 27 ET cases were identified. Meat consumption and meat cooking practices were compared to 200 controls. Detailed data were collected via questionnaires regarding current meat consumption for 14 meat types and meat cooking doneness for 8 meat types. Data were also available on blood harmane concentrations. Results: Current meat consumption was similar in ET cases and controls in 12 out of 14 meat types, with no differences observed after a Bonferroni correction in any meat type; no difference was observed when stratified by gender. No difference was observed in meat doneness between ET cases and controls. Blood harmane concentrations were not correlated with dietary data. Discussion: This is the first population-based study of harmane-linked dietary factors in ET. The study suggests the observed difference in blood harmane in ET is not driven by dietary differences and is likely due to other mechanisms (e.g., impaired metabolism).

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The simultaneous formation of acrylamide; ß-carboline heterocyclic amines (HAs): harmane and norharmane; and advanced glycation end products (AGEs) (Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL)) was analyzed based on an aqueous model system. The model systems included lysine-glucose (Lys/Glu), asparagine-glucose (Asn/Glu), tryptophan-glucose (Trp/Glu), and a mixture of these amino acids (Mix/Glu). Only AGEs were generated when heated at 100 °C, Asn and Trp competed with Lys for glucose and methylglyoxal (MGO), and glyoxal (GO) decreased AGE content. The k value of CML, CEL, and acrylamide decreased when heated at 130 °C, whereas that of harmane increased in the Mix/Glu, owing to the competition between Lys and Asn for glucose, GO, and MGO. Harmane preferably formed via the Pictet-Spengler condensation between Trp and acetaldehyde, which further reduced acrylamide formation via the acrolein pathway.

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In the present set of experiments, we studied the correlation between the heterocyclic amine (HCA) concentration and the color changes of the chicken breast with or without skin during grilling under open or closed conditions as a function of the applied temperature and time. The concentration of the HCAs formed during grilling was measured by a validated LC-MS/MS method, whereas the color changes were determined either instrumentally or by visual observation. In general, higher temperatures and longer heat treatment times resulted in a more substantial HCA formation, especially on the surface of the samples and in the skin, where the total levels reached 746 ng/g. Results of regression analysis demonstrate a strong correlation (r > 0.7) between the HCA content of the grilled chicken breast and the L* and a* values indicating the significance of brightness and the red parameter of the color scale, respectively. In the case of open grilling, the skinless breast samples showed correlation (r > 0.7) between the HCA content and the color analysis results in both the full sample and the crust, respectively. Breast samples with skin exhibited the same level of correlation when they were grilled closed. In the case of open grilling the breast with skin, and closed-grilling the skinless breast, the linear regression analysis yielded a weaker correlation (0.7 > r > 0.4 or less) between the HCA concentrations and the color. Our results demonstrate that there is a predictive correlation between the color changes perceptible for the consumers and the HCA formation during grilling of chicken breast as a function of time and temperature depending on the type of grilling and the presence of skin.

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BACKGROUND: Essential tremor (ET) is among the most prevalent neurological diseases. Its environmental determinants are poorly understood. Harmane (1-methyl-9H-pyrido[3, 4-b]indole), a dietary tremor-producing neurotoxin, has been linked to ET in a few studies in New York and Madrid. Mercury, also a tremor-producing neurotoxin, has not been studied in ET. The Faroe Islands have been the focus of epidemiological investigations of numerous neurological disorders. OBJECTIVE: In this population-based, case-control study, we directly measured blood harmane concentrations (HA) and blood mercury concentrations (Hg) in ET cases and controls. METHODS: In total, 1,328 Faroese adults were screened; 26 ET cases were identified whose (HA) and (Hg) were compared to 197 controls. RESULTS: Although there were no statistically significant differences between diagnostic groups, median (HA) was 2.7× higher in definite ET (4.13 g-10/mL) and 1.5× higher in probable ET (2.28 g-10/mL) than controls (1.53 g-10/mL). Small sample size was a limitation. For definite ET versus controls, p = 0.126. (Hg) were similar between groups. CONCLUSIONS: We demonstrated marginally elevated (HA) in definite and probable ET. These data are similar to those previously published and possibly extend etiological links between this neurotoxin and ET to a third locale. The study did not support a link between mercury and ET.

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Heterocyclic aromatic amines (HAAs) are mutagens and potential human carcinogens. Our group and others have demonstrated that HAAs may also produce selective dopaminergic neurotoxicity, potentially relevant to Parkinson's disease (PD). The goal of this study was to elucidate mechanisms of HAA-induced neurotoxicity through examining a translational biochemical weakness of common PD models. Neuromelanin is a pigmented byproduct of dopamine metabolism that has been debated as being both neurotoxic and neuroprotective in PD. Importantly, neuromelanin is known to bind and potentially release dopaminergic neurotoxicants, including HAAs (eg, ß-carbolines such as harmane). Binding of other HAA subclasses (ie, aminoimidazoaazarenes) to neuromelanin has not been investigated, nor has a specific role for neuromelanin in mediating HAA-induced neurotoxicity been examined. Thus, we investigated the role of neuromelanin in modulating HAA-induced neurotoxicity. We characterized melanin from Sepia officinalis and synthetic dopamine melanin, proposed neuromelanin analogs with similar biophysical properties. Using a cell-free assay, we demonstrated strong binding of harmane and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to neuromelanin analogs. To increase cellular neuromelanin, we transfected SH-SY5Y neuroblastoma cells with tyrosinase. Relative to controls, tyrosinase-expressing cells exhibited increased neuromelanin levels, cellular HAA uptake, cell toxicity, and oxidative damage. Given that typical cellular and rodent PD models form far lower neuromelanin levels than humans, there is a critical translational weakness in assessing HAA-neurotoxicity. The primary impacts of these results are identification of a potential mechanism by which HAAs accumulate in catecholaminergic neurons and support for the need to conduct neurotoxicity studies in systems forming neuromelanin.

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The occurrence of glucose-derived ß-carboline alkaloids tangutorid E (Tan E) and tangutorid F (Tan F) as well as their dehydroxy-derivatives (DH-Tan E/F) was investigated in a broad variety of foodstuffs by LC-MS/MS-based stable isotope dilution analysis (SIDA). For that purpose, the target compounds and their 13C6-stable isotope-labeled analogues were synthesized from l-tryptophan and (13C6-)d-glucose and used to develop a rapid LC-MS/MS-SIDA method. After validation for several food matrices, the method was applied to the analysis of these ß-carbolines in 80 food items. Quantitative amounts were detected in 46.3, 50.0, and 42.5% of the samples regarding Tan E, Tan F, and DH-Tan E/F, respectively, with corresponding ranges of 0.01-6.75, 0.01-5.07, and 0.01-0.75 mg/kg; the highest amounts were found in processed tomato products. A combination of the obtained occurrence data in foods with average food consumption data led to the calculation of rough estimates for the chronic daily intake of those alkaloids, yielding values of 0.44, 0.36, and 0.13 µg/kg body weight/day for Tan E, Tan F, and DH-Tan E/F, respectively. Evidently, the consumption of processed tomato-based products accounts for the majority of the total daily intake of the investigated ß-carbolines; the potential bioactivities of Tan E, Tan F, and DH-Tan E/F have yet to be investigated.

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Harmane, as a neuromodulator, implicates in the learning and memory processes. However, rapid eye movement (REM) sleep deprivation negatively affects these processes. Here, we investigated the effects of harmane (2.5 mg/kg) on the regulation of fear memory in free moving groups (FMG), large platform groups (LPG) and REM-deprived mice. We employed a flower pot technique for REM sleep deprivation and a Pavlovian fear conditioning paradigm for assessment of fear memories. FMGs received two or three pre-training intraperitoneal administrations of harmane at 12 h intervals, impaired contextual memory retention but those received three harmane administrations showed an auditory memory disruption. LPGs, with or without harmane, did not alter fear memories compared to their respective FMGs, indicating the inability of stress on fear responses of FMGs. Moreover, 12, 24 and 36 h REM sleep deprivation impaired contextual memory retrieval, while 24 and 36 h REM sleep deprivation impaired auditory fear memory retention. Furthermore, harmane only abolished contextual and auditory fear memory deficits induced by 24 h REM sleep deprivation. The data suggests a modulatory role for harmane in REM sleep deprivation response on fear memory.

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