bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generating Hypertrophic Adipocytes from 3T3 L1 Cell Line and Characterizing the Phenotypic Transition
Authors: Yuan Xinxu , Xu Xiaoyang , Li Ningjun , Dewey William L. , Li Pin‐Lan , Zhang Fan 
Published: November 2023

3T3 L1 is a classical preadipocyte cell line that has been extensively used for the study of adipogenesis, a physiological process giving rise to the adipocytes. Adipocytic hypertrophy, a pathological enlargement of adipocytes, underscores the occurrences of obesity‐associated metabolic syndrome. Currently, it is lack of a reliable cell model to simulate the hypertrophic transition of adipocytes. This study aims to develop adipocytic hypertrophy from 3T3 L1 and characterize the related phenotypic transition by examining the genes key to the pathophysiology of adipocytes as well as their functional outcomes. To generate hypertrophic adipocytes, we grew the post‐differentiated 3T3 L1 cells in DMEM/F12 medium (the starting date designated as day 0), supplemented with 10% FBS and 0.1% insulin, to their maturation approximately at day 10; and then continued loading lipids to the matured adipocytes by culturing these cells in the same media. The hypertrophic phenotype appeared around day 24. Morphologically, the further enlargement of lipid droplets during the hypertrophy was confirmed by Bodipy‐stained fluorescence images. Quantitative RT‐PCR analysis showed the expression of PPARγ, an adipogenesis‐promoting nuclear factor, and adiponectin, an adipogenetic indicator, were first progressively increased from day 0 to reach their maximum at the day 10, and then regressively declined to day 32, an end point in experimental observations. Consistent with the decline in adipogenesis, the mRNA levels of pro‐inflammatory factor MCP‐1 were markedly elevated in the hypertrophic adipocytes after day 24, as well, their MCP‐1‐containing culture media displayed significant macrophage‐attracting activity in cell migration assays. But the expression and activity of cytochrome C oxidase, a critical enzyme for energy generation, were reduced during the hypertrophy. Functionally, the viability of adipocytes, as determined by colorimetrically reading the formation of formazan from the metabolized tetrazolium, was gradually decreased after day 16. In situ TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) analysis showed the adipocyte apoptosis was significantly increased during the hypertrophy. These results for the first time demonstrate that hypertrophic adipocytes can be generated from 3T3 L1 cell line. And this hypertrophic transition may be able to serve as a model to develop adipocytic hypertrophy‐targeting remedy in the treatment of obesity‐associated metabolic syndrome.Support or Funding InformationSupported by NIH R01HL115068
Effects of Melatonin and Beta‐Amyloid on Mitochondrial Function in a Model of Aging Mouse Astrocytes
Authors: Debner Emily K , King Jonathan M 
Published: November 2023

BackgroundAstrocytes provide support for neurons, however, with age, astrocytes’ ability to provide neuroprotection decreases. This decrease in neuroprotection increases the likelihood of neurodegenerative diseases, that compromise function. One hallmark of neurodegeneration is accumulation of β‐amyloid (βA) plaques, which disrupts cellular function and often leads to cellular death. As astrocytes age, they have been shown to naturally have lower mitochondrial membrane potential (MMP) and more frequent with larger amplitude calcium waves. Treatment with melatonin has been shown to decrease βA production and prevent plaque formation in the brain, suggesting melatonin plays a role during neurodegeneration. The interplay between melatonin and βA effects on astrocyte function was examined by measuring MMP and calcium waves.MethodsPrimary mouse astrocyte cultures obtained from young (4 month) and old (28 month) animals were used in the current study. Astrocytes were cultured for 24hrs with 100μM melatonin before loading with either MitoID or Fluo3. Measures were obtained using galvano or resonant scanning modes on a confocal microscope system, respectively. Astrocytes were then treated with 0.5μM βA (AggreSure) and imaged for an additional 30 minutes. MMP and calcium waves were analyzed to indicate cellular stress and change in cellular function.ResultsMelatonin significantly elevated baseline MMP in young astrocytes while protecting MMP loss due to βA exposure. Baseline MMP levels were unaffected by melatonin in old astrocytes although melatonin preserved MMP levels during βA exposure. This suggests young astrocytes capacity to alter cellular activities due to melatonin exposure is more robust. Calcium waves in young astrocytes persisted for a longer duration during βA exposure.ConclusionsThese results indicate that melatonin has protective effects on preserving MMP in the presence of βA, but produced greater effects in younger astrocytes. These data indicate effects are related to cellular vitality and not due to accumulation of βA.Support or Funding InformationSupport:Trinity University Murchison Undergraduate Research Fellowship (EKD), Trinity University Biology Department and Neuroscience Program.
Novel Synthetic Cannabinoid AB‐PINACA Produces THC‐Like Behavioral and Cellular Effects Through activation of the Cannabinoid CB1 Receptor
Authors: Tai Sherrica , Crane Jasmine , Ford Benjamin M , Prather Paul L , Fantegrossi William E 
Published: November 2023

Novel synthetic cannabinoid AB‐PINACA has been identified in herbal products marketed as K2/Spice. Limited information exists on the affinity, intrinsic efficacy, or acute and repeated in vivo effects elicited by AB‐PINACA. In in vitro receptor binding studies, AB‐PINACA displayed high affinity at the CB1R where it functioned as a full agonist. In the tetrad assay, mice injected with 3 mg/kg AB‐PINACA exhibited hypothermia, antinociception, catalepsy, and hypolocomotion, all of which are classical cannabinoid agonist effects. Importantly, these effects were attenuated by rimonabant, a CB1R antagonist/inverse agonist, suggesting that AB‐PINACA mediates these behavioral effects through activation of the CB1R. Mice previously prepared with radiotelemetry probes capable of simultaneously monitoring core body temperature and locomotor activity were repeatedly administered 3 mg/kg AB‐PINACA once every 24 hrs for 5‐days. Mice repeatedly administered AB‐PINACA developed rapid and progressive tolerance to hypothermic effects, and this tolerance was still evident when re‐challenged with the same dose after a 14‐day drug abstinence period. However, no tolerance developed to hypolocomotion. Lastly, mice were administered 30 mg/kg THC repeatedly for 4 days. We have previously shown that this dose regimen produces downregulation and desensitization of CB1Rs and elicits tolerance to cannabinoid induced hypothermia. On Day 5 these mice were challenged with 3 mg/kg AB‐PINACA to determine cross‐tolerance. These data demonstrate that – similar to other high‐efficacy synthetic cannabinoid drugs of abuse – the intrinsic efficacy, acute and repeated in vivo effects of AB‐PINACA are similar to those of THC, and are mediated through agonist interactions with CB1Rs.Support or Funding InformationThis work was supported by RR029884, RR020146 and UAMS Department of Pharmacology and Toxicology.
Behavioral Characterization of Spiradoline and CP55,940 in Rats: Potential of Drug Mixtures for Treating Pain
Authors: Minervini Vanessa , France Charles Patrick 
Published: November 2023

Pain is a significant clinical problem and there is a need for drugs that are more effective with less adverse effects. Previous studies have shown that cannabinoid receptor agonists enhance the antinociceptive effects of mu opioid receptor agonists. It is unknown whether the well‐documented antinociceptive effects of kappa opioid receptor agonists are enhanced by cannabinoids and, therefore, whether kappa opioid receptor agonists, which are devoid of abuse liability, might be clinically useful in a drug mixture for treating pain. Cumulative dose‐effect functions (30‐min inter‐injection interval) were determined for the kappa opioid receptor agonist spiradoline (0.032 – 32.0 mg/kg) and the cannabinoid receptor agonist CP55,940 (0.0032 – 1.0 mg/kg) in 8 male Sprague Dawley rats for antinociception, schedule‐controlled behavior, body temperature, and diuresis. Spiradoline and CP55,940 dose dependently increased the latency for rats to remove their tails from warm (50°C) water to 100% of the maximum possible effect (20 s), while eliminating responding for food and decreasing body temperature by 5°C. CP55,940 was approximately 20‐ and 6‐fold more potent than spiradoline in the antinociception and the schedule‐controlled responding procedures, respectively. Spiradoline was 18‐fold and CP55,940 was 5‐fold more potent in the schedule‐controlled responding procedure than in the antinociception procedure. For both drugs, the rank order potencies across assays were schedule‐controlled responding > body temperature > antinociception. Spiradoline significantly increased (7‐fold) diuresis at doses that eliminated schedule‐controlled responding, whereas CP55,940 did not have a significant effect on urine output. These results provide the basis for examining mixtures of spiradoline and CP55,940 to test whether the preferred and potentially clinically useful effects of kappa opioid receptor agonists (antinociception) are selectively enhanced by cannabinoid receptor agonists.Support or Funding InformationThis work was supported, in part, by NIDA grants K05DA017918 and T32DA031115.
Daily Synthetic Cannabinoid Agonist (JWH‐018) Treatment: Tolerance to Δ9‐Tetrahydrocannabinol‐Like Discriminative Stimulus Effects in Rhesus Monkeys
Authors: Cocke Morgan L , McMahon Lance R 
Published: November 2023

Abused synthetic cannabinoids are used repeatedly, yet the consequences of repeated treatment on sensitivity to abuse‐related effects have not been established. This study assessed sensitivity to the discriminative stimulus effects of JWH‐018 and Δ9‐tetrahydrocannabinol (Δ9‐THC) before and immediately after daily JWH‐018 treatment in rhesus monkeys. Rhesus monkeys discriminated Δ9‐THC (0.1 mg/kg i.v.) under an FR5 schedule of stimulus‐shock termination. Cumulative dose‐response functions for JWH‐018 and Δ9‐THC were determined before and the day after 7 days of twice daily treatment with 0.32 mg/kg of JWH‐018 s.c. 6 h apart. Drug discrimination sessions were suspended during daily JWH‐018 treatment. Δ9‐THC and JWH‐018 dose‐dependently increased Δ9‐THC lever responding; the respective ED50 values were 0.017 mg/kg and 0.0077 mg/kg. After daily JWH‐018 treatment, the ED50 values of JWH‐018 and Δ9‐THC were significantly increased, as evidenced by potency ratios (95% confidence limits) of 4.1 (2.1–8.1) and 6.6 (5.0–8.8), respectively. The magnitudes of increases in ED50 value were not significantly different between JWH‐018 and Δ9‐THC. Twice daily vehicle treatment for 7 days did not significantly alter sensitivity to JWH‐018, i.e., the potency ratio (95% confidence limits) comparing the ED50 value after vs. before vehicle treatment was 0.4 (0.2–1.2). Full substitution of JWH‐018 for Δ9‐THC, coupled with the similar tolerance to JWH‐018 and cross‐tolerance to Δ9‐THC as a function of daily JWH‐018 treatment, are consistent with a common pharmacological mechanism. Daily synthetic cannabinoid agonist use can result in tolerance and a likely need to increase the frequency and quantity of use to maintain subjective effects.Support or Funding InformationUSPHS grant R01 DA19222 and T32 NS082145
Comparison of Heroin‐ and Δ9‐Tetrahydrocannabinol‐Induced Antinociception and Physical Dependence in Monkeys
Authors: Ding Huiping , Kiguchi Norikazu , Kishioka Shiroh , Ko Mei‐Chuan 
Published: November 2023

Both the opioid heroin and the cannabinoid Δ9‐Tetrahydrocannabinol (Δ9‐THC) exhibit analgesic effects, but both drugs produce undesirable abuse liability. Abuse of opioids and cannabis is a serious global problem that affects all societies on multiple levels. However, little is known about the development of physical dependence on these two classes of drugs in primates. Thus, the aim of this study was to compare the potency of heroin‐ and Δ9‐THC‐induced analgesic effects and the development of physical dependence on these two drugs in rhesus monkeys. Systemic administration of either heroin (0.03–0.18 mg/kg) or Δ9‐THC (0.3–1.8 mg/kg) dose‐dependently produced antinociceptive effects against acute thermal nociceptive stimulus. The opioid receptor antagonist naltrexone (0.01 mg/kg) and the cannabinoid receptor antagonist SR141716A (0.3 mg/kg) produced the same degree of a rightward shift of the dose‐response curve for heroin‐ and Δ9‐THC‐induced antinociception, respectively. To study acute physical dependence on heroin and Δ9‐THC, monkeys were subjected to a short‐term repeated administration (two injections per day for 2 to 3 days) of either heroin (0.18 mg/kg) or Δ9‐THC (1.8 mg/kg). Then, monkeys were treated with either naltrexone (0.01 mg/kg) or SR141716A (0.3 mg/kg), i.e., antagonist‐precipitated withdrawal. We found that surgically implanted telemetric probes (DSI, model D70‐PCTR) are sensitive in detecting physiological withdrawal signs. Administration of naltrexone increased heart rate and blood pressure in heroin‐treated monkeys, indicating a fast development of physical dependence on heroin. Depending upon the duration of repeated administration of Δ9‐THC and the dose of SR141716A, development of physical dependence on Δ9‐THC can be detected too. Collectively, these results demonstrate that in nonhuman primates, systemic heroin is more potent than Δ9‐THC in producing antinociception, and physical dependence on heroin can occur following a short‐term exposure.Support or Funding InformationSupported by US‐PHS grants DA‐032568 and DA‐035359.
Impact of Chronic Ethanol Self‐Administration on Kappa Opioid Receptor Regulation of Dopamine Signaling in Nonhuman Primates
Authors: Siciliano Cody A , Calipari Erin S , Fordahl Steven C , Melchior James R , Yorgason Jordan T , Mateo Yolanda , Helms Christa M , Jimenez Vanessa A , Lovinger David M , Grant Kathleen A , Jones Sara R 
Published: November 2023

Although alcoholism is one of the most prevalent disorders in the United States, with over 18 million individuals meeting the criteria for an alcohol use disorder, the exact neurobiological bases of this condition remain obscure. Recently, it has been demonstrated that kappa‐opioid receptor (KOR) signaling in the striatum plays a critical role in the increased reinforcing efficacy of ethanol following ethanol vapor exposure in rodent models. Here we examined the effects of chronic voluntary ethanol self‐administration in macaques on dopamine neurotransmission and the ability of KORs to regulate dopamine release in the nucleus accumbens core. Three cohorts of nonhuman primates were given free access to 4% ethanol (w/v) for 22 hr/day. These cohorts were composed of male cynomolgus, female rhesus or male rhesus macaques, and were given access to ethanol for 6, 12, or 18 months, respectively. Ex vivo fast‐scan cyclic voltammetry was then conducted the nucleus accumbens core to determine dopamine signaling kinetics as well as the ability of U50,488 (KOR agonist) to inhibit dopamine release. We found that chronic ethanol drinking increased dopamine uptake rates, which could have implications for reductions in basal dopamine tone in vivo during ethanol withdrawal. Further, across sex, strain and exposure length, ethanol use augmented the ability of KORs to inhibit dopamine release, demonstrating that ethanol‐induced increases in KOR sensitivity are widespread and independent of other factors. Finally, KOR sensitivity was positively correlated with lifetime ethanol intake, suggesting that changes in KOR regulation of dopamine release may be a determinant of aberrant drinking behaviors. Nonhuman primate models of ethanol abuse represent a highly translational avenue for identifying molecular targets for pharmacotherapeutic compounds, and here we show, for the first time, that voluntary ethanol self‐administration has a unique effect on KOR sensitivity and regulation of dopamine release directly at the dopamine terminal that was positively correlated with drinking behavior. Together, these data provide novel insight into ethanol‐induced dysregulation of opioid signaling and suggest that dopaminergic dysfunction may be mediating increases in voluntary drinking. Importantly, KOR antagonists may be efficacious in reducing drinking behaviors in alcoholics.Support or Funding InformationThis work was funded by NIH grants U01 AA014091, P01 AA021099 (SRJ), F31 DA031533 (ESC), F31 DA037710, T32 AA007565 (CAS), F31 AA020439 (JTY), P51 OD011092, R24 AA019431, P60 AA10760 (KAG), Division of Intramural Clinical and Biomedical Research NIAAA (DML), Integrative Neuroscience Initiative on Alcoholism AA 13510 (KAG).
The Serotonin 2A Receptor Agonist 2,5‐dimethoxy‐4‐iodoamphetamine (DOI) Reduces Ethanol Consumption and Place Conditioning
Authors: Murnane Kevin Sean , Nakhla Peter , Curry Kristen , Phillips John , Daphney Cedrick 
Published: November 2023

17 million adults in the United States ages 18 and older had an alcohol use disorder (AUD) in 2012. Current FDA‐approved treatments for AUD are limited to disulfiram, naltrexone, and acamprosate, and many patients are refractory to these treatments. Hallucinogenic drugs were used in the 1960s to treat AUD, and a number of studies supporting their use were published. Moreover, recent studies have documented impressive effects of psilocybin for tobacco cessation and ayahuasca for the prevention of ethanol‐induced behavioral sensitization. Therefore, herein, we investigated the effects of the serotonin 2A receptor agonist and hallucinogen 2,5‐dimethoxy‐4‐iodoamphetamine (DOI) in ethanol‐induced place conditioning and 2‐bottle choice ethanol consumption in mice, 2 widely accepted animal models of ethanol abuse. We found that DOI pretreatment prevented the development of an ethanol‐induced place preference, and moreover, DOI treatment after ethanol prevented the consolidation of such conditioning. DOI treatment robustly decreased ethanol consumption without affecting total fluid intake, and interestingly, this effect was most profound in subjects that exhibited high ethanol consumption. As there are clear clinical limitations to the use of hallucinogenic drugs, we went on to explore whether computational chemistry approaches could be used to develop biased agonists of serotonin 2A receptors with reduced hallucinogenic activity, and whether such compounds suppressed ethanol consumption. These studies document a new model system in which we can now investigate the neurobiological and pharmacological mechanisms through which hallucinogens reduce ethanol consumption and appetitive conditioning.Support or Funding InformationSupport was provided by Mercer University and the Mercer University College of Pharmacy.
Tianeptine Co‐administration with Alcohol to Adolescent Rats Modifies the Alcohol Withdrawal Syndrome
Authors: Rhoads Dennis E. , Martin Bryan , Mauterer Madelyn , Orlando Krystal 
Published: November 2023

Adolescence is a time when many individuals have their first experiences with alcohol and other drugs. It is a time when the brain undergoes important developmental changes that may be altered by drug use. We have shown previously that adolescent Long‐Evans rats consume high levels of an alcohol‐containing liquid diet, achieve correspondingly high blood alcohol concentrations, and develop a subsequent severe alcohol withdrawal syndrome. Using adult rats, tianeptine has been shown by other groups to decrease anxiety‐like behaviors and the onset and severity of convulsions during alcohol withdrawal. The present study was designed to test the effectiveness of tianeptine against the severe alcohol withdrawal syndrome seen with adolescent rats and to test the effects of tianeptine on upregulation of glutamate receptors during adolescent alcohol consumption. Liquid diets were used to administer alcohol and tianeptine, alone and in combination, using a modified chronic intermittent design. After 10 days of co‐administration of tianeptine and alcohol and 6 hours of withdrawal, motor activity showed little or none of the hypoactivity characteristic of alcohol withdrawal. Tianeptine alone had no effect on motor activity under these conditions. However, continued out to 21 days of co‐administration, adolescent rats showed the same high frequency of induced running episodes and convulsions during withdrawal as when alcohol was administered alone. Western blotting was used to determine expression levels for forebrain glutamate receptors, AMPA and NMDA. Both receptors were upregulated during chronic alcohol consumption. During co‐administration with alcohol, tianeptine delayed this upregulation so that at 10 days there was no significant upregulation of AMPA or NMDA whereas significant increases in expression were observed by 21 days. These results indicate that tianeptine may dampen symptoms of adolescent alcohol withdrawal in the short term by delaying the upregulation of excitatory glutamate receptors induced by chronic alcohol. However, tianeptine was ineffective in maintaining this delay and in preventing more severe withdrawal associated with longer term alcohol consumption. Thus, we conclude that tianeptine may be of limited effectiveness against alcohol withdrawal severity in adolescents compared to that suggested previously for adult rats.Support or Funding InformationThis work was supported by the Biology Department and School of Science of Monmouth University.