bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of opioid and serotonergic systems by alpha‐terpineol complexed in beta‐cyclodextrin reduces hyperalgesia in a chronic muscle pain model
Authors: Guimarães Adriana Gibara , Oliveira Makson Gleydson Brito , Menezes Paula Passos , de Souza Araújo Adriano Antunes , Silva Juliane Cabral , da Silva Almeida Jackson Roberto Guedes , Santos Márcio Roberto Viana , Júnior Lucindo José Quintans
Published: November 2023

The aim of this study was to evaluate the effect of α‐terpineol, an alcohol monoterpene found in the essential oil of aromatic species, complexed in β‐cyclodextrin (TP‐βCD) in chronic muscle pain noninflammatory model and its mechanism of action. The model of chronic muscle pain was induced by two injections of pH 4.0 saline (20 μl) into the left gastrocnemius 5 days apart. After confirming hyperalgesia, male mice were treated with α‐terpineol (αTPN 100 mg/kg, p.o.), TP‐βCD (25, 50 or 100 mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) daily for 10 days. The possible involvement of the opioid and serotonergic systems were evaluated using formalin test (1%; 20 μL; i.pl.), which animals were pretreated with αTPN‐βCD (p.o.) or vehicle (p.o.), naloxone (an opioid antagonist, 5 mg/kg; i.p.) or ondansetron (an 5HT3 antagonist, 0.5 mg/kg; i.p.). The experimental protocols were approved for the UFS Ethic Committee (CEPA: 08/11). TP‐βCD reduced significantively (p<0.001) the mechanical hyperalgesia when compared with vehicle. Besides, the complexation with βCD increased the effect time of TP of 3 hrs to 7 hrs. Naloxone and ondansetron were able to reverse this analgesic effect. These results demonstrate that TP‐βCD reduced the hyperalgesia followed by the chronic muscle pain model, probably evoked by the descending inhibitory pain system, specifically by opioid and serotoninergic receptors.

Support or Funding Information

FAPITEC/SE, CAPES, CNPq (Brazil).
Characterization of the Ability of Diazepam to Reverse Prescription Opioid Tolerance
Authors: Gonek Maciej , Henderson Graeme , Dewey William L.
Published: November 2023

We are experiencing an unprecedented opioid overdose epidemic with over 120 people dying every day. Most individuals who become tolerant and dependent on opioids, including the prescription opioids oxycodone and hydrocodone, co‐abuse other substances. The most widely co‐abused drug is ethanol but other CNS depressants such as diazepam and other benzodiazepines are also co‐abused. Several studies suggest that ethanol and benzodiazepines may play a role in as much as 80% of unintentional overdose deaths involving opioids. Postmortem examination of individuals who have died from chronic administration of opioids and ethanol had lower blood levels of opioids than individuals who overdosed on opioids alone. Similar results have not been reported in individuals co‐abusing diazepam. Following the hourly subcutaneous administration of the acute ED80 dose for a total of 7 injections, we observed a significant shift of the dose response curve indicating a 5‐fold tolerance to morphine, a 1.5‐fold tolerance to oxycodone, and a 2‐fold tolerance to hydrocodone in the mouse tail immersion assay. The intraperitoneal administration of 0.5 mg/kg diazepam, a dose that has no observable behavioral effect on its own, 50 minutes before testing fully reversed antinociceptive tolerance to morphine and oxycodone. Hydrocodone tolerance was not fully reversed at doses lower than 2 mg/kg diazepam. There was no potentiation of acute opioid effects. Conversely, opioid tolerance in the stretching assay, a measurement of visceral pain, was not reversed by diazepam. Further research into the differences of opioid tolerance mechanisms can lead to new insights for the development of drugs that decrease the risk of opioid overdosing and the development of drugs to relieve severe pain with limited tolerance.

Support or Funding Information

Supported by USPHS Grants RO1 DA036975 and T32DA007027.
Abuse Liability and Anti‐Addiction Potential of the Atypical Mu Opioid Receptor Agonist IBNtxA
Authors: Keck Thomas M , Uddin Md. Main , Babenko Elvira , Wu Chun , Moura‐Letts Gustavo
Published: November 2023

OBJECTIVE

IBNtxA is a μ opioid receptor (MOR) agonist structurally related to the classical MOR antagonist naltrexone. Recent studies suggest IBNtxA preferentially signals through truncated MOR splice variants, producing a unique pharmacological profile resulting in potent analgesia with reduced side effects, including no conditioned place preference (CPP) when tested at a single dose. The purpose of this study is to 1) evaluate a range of IBNtxA doses to more fully assess its abuse liability and 2) determine the effects of IBNtxA on morphine CPP expression and reinstatement of morphine CPP.

METHODS

IBNtxA was synthesized and compared to morphine in standard CPP expression assays. Following morphine CPP training, IBNtxA was tested for its effects in inducing CPP reinstatement on its own or attenuating morphine‐primed reinstatement. Drug discrimination studies are also underway.

RESULTS and CONCLUSIONS

IBNtxA represents an intriguing lead compound for preclinical drug development specifically targeting MOR splice variants, potentially creating effective analgesics with reduced side effects. Furthermore, IBNtxA could have use as an adjunct therapy in agonist replacement strategies (e.g., methadone). Current collaborative efforts are aimed at developing novel analogues of IBNtxA for further analysis and understanding ligand‐receptor interactions across MOR splice variants using molecular modeling.

Support or Funding Information

Rowan University Seed Fund; travel support from Dean of Rowan University College of Science and Mathematics.
Preference for an Opioid/Benzodiazepine Mixture over an Opioid Alone using a Concurrent Choice Procedure in Rhesus Monkeys
Authors: Weed Peter Fletcher , France Charles , Gerak Lisa
Published: November 2023

Opioid abuse has become an epidemic, leading to a dramatic increase in emergency room visits and overdose deaths. Opioids are frequently coabused with other drugs, and up to 70% of opioid abusers report concurrent benzodiazepine abuse. Coabuse of opioid/benzodiazepine mixtures might be due to benzodiazepines enhancing the positive‐reinforcing effects (increasing potency and/or effectiveness) of opioids. The current study examined whether monkeys choose to self‐administer a mixture of the opioid remifentanil and the benzodiazepine midazolam over remifentanil alone. Three monkeys responded under a concurrent choice procedure for intravenous drug infusions. Initially remifentanil or midazolam was available when the alternative was saline. After dose‐response curves for remifentanil and midazolam were established, monkeys could choose between a dose of remifentanil (0.32 μg/kg/infusion) that did not change during the study and a dose of remifentanil that varied across sessions; for some sessions, midazolam was combined with varying doses of remifentanil. When saline was the alternative, the rate at which monkeys responded for infusions of remifentanil (0.0032 – 0.32 μg/kg/infusion) increased dose‐dependently with all monkeys earning most or all of the available infusions at the largest dose whereas the rate at which monkeys responded for infusions of midazolam increased modestly in only one subject at one dose (10 μg/kg/infusion). When 0.32 μg/kg/infusion of remifentanil was available on one lever and a dose of remifentanil that varied across sessions (0.1 – 1 μg/kg/infusion) was available on the other lever, monkeys chose the larger dose. Combining a small dose of midazolam (3.2 μg/kg/infusion) with 0.32 μg/kg/infusion of remifentanil increased responding for the mixture over 0.32 μg/kg/infusion of remifentanil alone; however, when smaller doses of remifentanil were combined with 3.2 μg/kg/infusion of midazolam, monkeys chose the larger dose of remifentanil alone. In contrast, monkeys chose 0.1 μg/kg/infusion of remifentanil when combined with 10 μg/kg/infusion of midazolam over a larger dose (0.32 μg/kg/infusion) of remifentanil alone. Thus, monkeys prefer some opioid/benzodiazepine mixtures over an opioid alone, suggesting that coabuse of opioid/benzodiazepine mixtures might be due to increased potency (and possibly effectiveness) of the reinforcing effects of opioids.

Support or Funding Information

Supported by USPHS grants R21DA039394 and T32DA031115 and the Welch Foundation Grant AQ‐0039.
Alterations in Nucleus Accumbens Dopamine Terminals Following Dopamine Tolerance‐ or Sensitization‐Inducing Cocaine Self‐Administration Protocols
Authors: Mauterer Madelyn I , Albertson Steven E , Jones Sara R
Published: November 2023

Cocaine has diverse effects on the function of the mesolimbic dopamine system, depending on the schedule and timing of self‐administration behavior. In cocaine self‐administering rats, sensitization as well as tolerance are phenomena that are not only observable behaviorally, but also at the molecular level, specifically at the dopamine transporter (DAT). When rats are allowed long access to cocaine (LgA), in which they can receive an unlimited number of infusions (1.5 mg/kg, i.v.) over the course of six hours per day for 14 days, cocaine intake moderately escalates and the DAT shows robust tolerance to cocaine in vitro. In a short access paradigm (ShA), during which rats have unlimited access to cocaine but only for two hours per day for 14 days, cocaine intake is constant and the DAT is unchanged, showing cocaine responsiveness that is the same as their drug naïve counterparts. However, in contrast, when rats are allowed intermittent access to cocaine (IntA), during which rats are allowed unlimited access to cocaine with no time‐outs for 5 minutes out of every 30 minutes, six hours per day for 14 days, cocaine intake during the 5 min bout is drastically increased and the DAT shows sensitization to the effects of cocaine in vitro. DAT sensitization occurs even though the animals undergoing the IntA paradigm have the same levels of cocaine intake as animals on the ShA paradigm. While the consequences of these self‐administration paradigms have been well characterized at the DAT, because opposite DAT changes occur with two schedules that produce escalation in dopamine‐dependent cocaine self‐administration behaviors, it is clear that there are other neurobiological changes contributing to these behaviors. It is not known how LgA, ShA, and IntA affect other aspects of the dopamine terminal, and so the purpose of this study is to characterize additional regulatory mechanisms on dopamine terminals. Using voltammetry in brain slices, we found that after an LgA protocol, there are no differences in the sensitivity of D2 or D3 dopamine release‐inhibiting autoreceptors compared to control animals. This juxtaposes previous findings from our lab that showed that autoreceptors became subsensitive following a limited access 24 hour paradigm. We are interested in the effects of IntA cocaine self‐administration on autoreceptor function, and the regulation of the strongly inhibitory kappa opioid receptors that are on dopamine terminals. By studying how other dopamine‐regulating processes, other than the DAT, respond to different self‐administrations paradigms, we will be able to gain a better understanding of how cocaine affects the dopamine system as a whole.

Support or Funding Information

R01DA014030

R01DA011697

T32AA007565
Assessment of the Specificity of the Effects of Rimcazole on the Reinforcing Effects of Cocaine Using a Behavioral Economic Approach
Authors: Job Martin O. , Katz Jonathan L.
Published: November 2023

Previous research has shown that cocaine self‐administration is decreased by rimcazole, a sigma (σ) receptor antagonist that also binds to the dopamine transporter (DAT). However, it is uncertain if those decreases are due to an action of rimcazole on the reinforcing effect of cocaine. One way to assess reinforcing effects is with an analysis of demand curves that show the decreases in consumption of a reinforcer with increasing cost. From the demand curve, the fitted parameters for consumption at no cost (Q0) and the essential value of the reinforcer (EV, a measure derived from the sensitivity to price) can be derived. Because rimcazole has actions at both the DAT and σ receptors, its effects were compared to that of combinations of a σ receptor antagonist (BD 1008) and a standard DAT inhibitor (methylphenidate). Rimcazole (3.2, 10 and 32 mg/kg) or vehicle were administered (i.p.) to male Sprague‐Dawley rats 5 minutes before sessions in which the number of lever presses required per infusion for either of two doses of cocaine (0.1 or 0.32 mg/kg/infusion) or food (three 20 mg pellets) were increased within sessions. The effects of combinations of methylphenidate (1 mg/kg i.p.) and different doses of BD 1008 (0, 3.2, 10 mg/kg) on the demand curve for cocaine (0.1 or 0.32 mg/kg/infusion) or food were determined under similar conditions. Demand curves were fitted to the data using an exponential model (Hursh and Silberberg, Psyc Rev 115: 186, 2008). Rimcazole dose‐dependently decreased both Q0 and EV at doses that had no effect on food‐reinforced responding. The combination of methylphenidate and the highest dose of BD 1008 also decreased Q0, though only at the 0.1 mg/kg/infusion dose of cocaine. In contrast the combination had no effect on the behavioral economic parameters derived from food‐reinforced responding. The present results indicate that rimcazole specifically alters the reinforcing effects of cocaine, but not food. Further, the similarity in effects of the combinations of methylphenidate and BD 1008 to rimcazole suggests that dual inhibition of both the DAT and σ receptors shows specificity in decreasing the reinforcing effects of cocaine and may provide targets for discovery of a combination chemotherapy for cocaine abuse.

Support or Funding Information

This work was supported by NIDA/NIH.
PKCβ Inhibitors Decrease AMPH‐Stimulated Behaviors: More than One Mechanism?
Authors: Altshuler Rachel D , Carpenter Colleen A , Gnegy Margaret E , Jutkiewicz Emily M
Published: November 2023

Amphetamines (AMPH) elicit their reinforcing effects by increasing dopamine (DA) release from neurons extending from the ventral tegmental area (VTA) to the nucleus accumbens (NAc). Protein kinase Cβ (PKCβ) has been shown to be important for AMPH's mechanism of action, such that inhibition of PKCβ reduces AMPH‐stimulated extracellular DA levels in vitro. While the effects of PKC inhibitors on AMPH‐stimulated DA efflux have been characterized, it is important to understand the effects of PKCβ inhibition in vivo. We previously showed that intraventricular (icv) injections of selective PKCβ inhibitors (ruboxistaurin or enzastaurin) decreases AMPH stimulated locomotor activity, AMPH self‐administration, and AMPH‐stimulated PKC activity but only when given 18 hours before AMPH. The goal of this study was to determine why PKCβ inhibitors given icv require a long pretreatment time before decreasing AMPH behaviors. Our hypothesis is that the site of action of a PKCβ inhibitor, the NAc (site of DA terminals) and VTA (site of DA cell bodies), determines the PKCβ inhibitors' time course. 10 pmols of ruboxistaurin were administered into the NAc or VTA of male Sprague‐Dawley rats 30 mins or 18 hrs prior to evaluating locomotor activity in response to 1 mg/kg AMPH (s.c.). Ruboxistaurin administered directly into the NAc with a 30 min, but not 18 hr, pretreatment decreased AMPH‐stimulated locomotor activity. Conversely, ruboxistaurin given directly into the VTA following an 18 hr, but not 30 min, pretreatment blocked AMPH‐stimulated locomotor activity. Therefore, acute inhibition of PKCβ in the NAc can decrease AMPH‐stimulated activity, but this effect is not observed following icv administration. This may suggest that following icv injections, the concentrations of the PKCβ inhibitor in the NAc are not large enough to affect locomotor activity acutely. The longer pretreatment time necessary for intra‐VTA PKCβ inhibitors to block AMPH‐stimulated behaviors suggests the involvement of a different mechanism that may be less reliant on direct PKC inhibition, such as alterations in transcription or protein synthesis. Overall, we conclude that PKC inhibition decreases AMPH‐stimulated behaviors through a direct and indirect mechanism. Future work will investigate the indirect mechanism, determining if PKCβ inhibitors given directly into the VTA alter PKCβ or DAT levels or function in the NAc.

Support or Funding Information

Funded by DA11697 and T32‐GM007767.
Effects of Daily Oral Phendimetrazine on Cocaine‐Food Choice in Rhesus Monkeys
Authors: Stowe Taylor Ashley , Nader Michael , Czoty Paul
Published: November 2023

Current treatments for cocaine abuse have not been efficacious in the majority of patients. One strategy examined in these studies is for pharmacotherapies to involve combination treatments in which one drug is a long‐acting “agonist” and a second drug administered in combination to perhaps enhance the effects of both drugs. Phendimetrazine is a clinically available anorectic agent that is a prodrug to phenmetrazine, an amphetamine‐like compound, and may have lower abuse liability in comparison to other agonist therapies. Research has shown phendimetrazine reduced cocaine choice by rhesus monkeys when given intravenously by continuous infusion (Banks et al., 2013). The present study examined the effects of oral phendimetrazine on cocaine choice in rhesus monkeys; this treatment route may better represent how the drug would be administered clinically. In this study, rhesus macaques (n=4) were trained to choose between banana‐flavored pellets and cocaine doses (0, 0.003–0.1 mg/kg/injection). Complete cocaine dose‐response curves were determined each session and cocaine choice increased as a function of cocaine dose. Once the monkeys' responding was stable, they were given phendimetrazine orally twice a day and tested once a week on the choice procedure. The dose of phendimetrazine always started at 4.0 mg/kg and increased depending on whether the monkey's choice changed. If the phendimetrazine dose lowered cocaine choice, the monkey would continue on this dose for another week. However, if cocaine choice remained at baseline or increased the dose of phendimetrazine would increase. Blood samples were taken weekly to ensure that the monkeys were taking the oral phendimetrazine. In preliminary findings, oral (6.0 mg/kg) phendimetrazine initially decreased cocaine choice in two monkeys, but tolerance developed to this effect. At present, it is unclear why these results vary from Banks et al. (2013), but could be due to a number of reasons such as the route and duration of administration of phendimetrazine or the availability of cocaine in which monkeys in the present study were tested weekly rather than daily in the Banks et al. study. Measures of plasma phendimetrazine and phenmetrazine will provide important information. Future directions include examining these methodological issues and to add a second drug to extend the initial effects of phendimetrazine in lowering cocaine choice.

Support or Funding Information

DA06634
Mephedrone: a Synthetic Cathinone with Marked Serotonergic Discriminative Stimulus Effects in Rats
Authors: Erwin Laura Lynn , Jursic Bronco , Winsauer Peter
Published: November 2023

Mephedrone (4‐methylmethcathinone) was one of the major constituents of the recreational substances referred to as “bath salts”. It is a synthetic cathinone and psychostimulant that can produce auditory and visual hallucinations, as well as problematic cardiovascular effects. This study compared the discriminative stimulus effects of mephedrone (0.32–10 mg/kg) with other prototypical drugs with central nervous system effects, such as ketamine (1.8–18 mg/kg), methylphenidate (1.0–10 mg/kg), and 3,4‐methylenedioxyamphetamine (MDA) (0.32–5.6 mg/kg). Additionally, a selective serotonin reuptake inhibitor, fluoxetine, was administered alone and in combination with mephedrone in order to assess the involvement of the serotonin transporter (SERT). Rats (n=11) were trained to discriminate an intraperitoneal injection of mephedrone from saline under a fixed‐ratio 20 schedule of food presentation. Following training, increasing cumulative doses of mephedrone (0.32–10 mg/kg) produced dose‐dependent increases in mephedrone‐lever responding, with full substitution considered to be greater than 80% drug‐lever responding and partial substitution greater than 50% drug‐lever responding. During substitution tests, MDA and methylphenidate produced 77% and 56% drug‐lever responding, respectively, without significantly decreasing rate of responding (i.e., a 20% decrease from control). Ketamine never produced more than 17% drug‐lever responding at any dose tested. When cumulative doses of fluoxetine (1.8–18 mg/kg) were administered alone, there was little to no substitution. However, when given as a pretreatment, 3.2 mg/kg fluoxetine in combination with mephedrone produced a small (less than two‐fold) shift to the right in the dose‐effect curve for mephedrone‐lever responding. As the pretreatment dose of fluoxetine increased to 5.6 and 10 mg/kg, the ascending portion of the dose‐effect curve for drug‐lever responding shifted to the left and upward. In addition, the 5.6 and 10 mg/kg doses of fluoxetine both partially substituted when administered alone as a bolus injection prior to mephedrone, something which was not seen when administered in cumulative doses. Taken together, these data indicate that the discriminative stimulus effects of mephedrone partially overlap with stimulants such as methylphenidate and MDA, and are also mediated by SERT due to the capacity of fluoxetine to dose‐dependently shift the ascending portion of the curve for mephedrone's discriminative stimulus effects. These data also suggest that doses in the range of 3.2–10 mg/kg likely involve other neurotransmitters such as dopamine.

Support or Funding Information

Board of Regents Fellowship
Dietary supplementation with fish oil prevents high fat diet induced enhancement of sensitivity to the behavioral effects of quinpirole in rats
Authors: Hernandez‐Casner Caroline , Ramos Jeremiah , Hussain Samirah , Serafine Kathrine M.
Published: November 2023

Eating a high fat diet can lead to negative health consequences, such as obesity and insulin resistance. Eating a high fat diet also enhances sensitivity of rats to the behavioral effects of drugs acting on dopamine systems, including the dopamine D2/D3 receptor agonist, quinpirole. To test the hypothesis that dietary supplementation with fish oil prevents high fat diet‐induced enhanced sensitivity to the behavioral effects of quinpirole (0.0032–0.32 mg/kg), adolescent male rats ate standard laboratory chow (17% kcal from fat), high fat chow (60% kcal from fat), standard chow supplemented with fish oil or high fat chow supplemented with fish oil. Similar to previous reports, after 5 weeks, rats eating high fat chow were more sensitive (e.g., leftward shift of the quinpirole dose‐response curve) than rats eating standard chow, to yawning induced by quinpirole. Dietary supplementation with 20% (w/w) fish oil prevented this effect. That is, quinpirole dose‐response curves were not different between rats eating high fat chow supplemented with fish oil and standard chow‐fed controls. These results suggest that although a high fat diet causes enhanced drug sensitivity to the behavioral effects of quinpirole, fish oil supplementation can prevent this. Further research is being conducted to understand the mechanisms underlying this effect, and to examine whether fish oil will reverse the effects of eating a high fat diet once they have already developed.
Effects of Alpha‐2 Norepinephrine Receptor Agonists on Cocaine Self‐Administration in Nonhuman Primate Models
Authors: Czoty Paul W , Coller Michael , Odom Jill , Epperly Phillip , John William S , Nader Susan H , Nader Michael A
Published: November 2023

Preclinical research has shown that drugs acting at alpha‐2 norepinephrine receptors can decrease stress‐induced reinstatement of cocaine self‐administration in rodents and stress‐induced cocaine craving in humans. Because stress can drive cocaine use, alpha‐2 receptor agonists such as lofexidine (LFX) may have utility in treating cocaine use disorder. In the only published study in nonhuman primates, acute LFX decreased cocaine self‐administration under a fixed‐ratio (FR) schedule, but chronic LFX treatment increased the reinforcing potency of cocaine (Kohut et al., Drug Alcohol Depend, 2013). To expand the range of conditions under which LFX has been studied, we examined effects of acute administration of LFX (0.03–3.0 mg/kg, i.v.) under several procedures in monkeys. More specifically, we examined LFX under conditions used to assess the reinforcing strength of cocaine using concurrent (choice) and progressive‐ratio (PR) schedules of reinforcement. Group‐housed male (n=8) and female (n=4) cynomolgus monkeys self‐administered cocaine under a food‐drug choice procedure. Under this paradigm, ascending cocaine doses (0.0, 0.003, 0.01, 0.03, 0.1 mg/kg per i.v. injection) were available as the alternative to a food pellet under a concurrent fixed‐ratio schedule in successive components of each daily session. Thus, a complete cocaine dose‐effect curve was determined each day. Under these conditions, LFX decreased choice of at least one cocaine dose in 5 of 8 male monkeys and 2 of 4 females; other monkeys were unaffected. In male monkeys, another alpha‐2 receptor agonist, guanfacine (0.01–1.0 mg/kg, i.v.), produced similar effects. In contrast, in 3 male rhesus monkeys, acute and chronic treatment with LFX increased choice of low cocaine doses. In addition, in 5 rhesus monkeys responding under a multiple FR food, PR cocaine schedule, LFX increased cocaine injections at doses that did not affect food‐reinforced responding. Taken together, these data indicate a limited effectiveness of alpha‐2 receptor agonists to decrease ongoing cocaine self‐administration. However, the potential of this class of drugs to reduce relapse in abstinent individuals should be studied further in models that specifically assess such effects.

Support or Funding Information

DA10584, DA 17763, DA 06634
Prior Treadmill Exercise Promotes Resilience in Traumatized Rats
Authors: Kochi Camila , Zaidi Safiyya , Liu Hesong , Atrooz Fatin , Dantoin Phoebe , Salim Samina
Published: November 2023

Post‐traumatic stress disorder (PTSD) is a serious psychological condition induced by experiencing traumatic events. Interestingly, PTSD can be acquired not only from directly experiencing traumatic events but also from witnessing such events. Treatment and prevention strategies are limited. Animal studies have provided critical insights. Recently, we published a “Trauma Witness Model” (TWM) of rat and reported that witnessing traumatic events cause PTSD‐like behaviors including anxiety‐ and depression‐like behavior as well as learning‐memory deficits. TWM is a modified social defeat paradigm, which involves aggressive encounters between a large Long‐Evans male rat (resident) and a smaller Sprague‐Dawley male rat (intruder), resulting in intruder social defeat. The cage mate of the intruder designated as trauma witnessing rat (TW), is positioned to witness intruder defeat. Using this model, we examined beneficial role of moderate treadmill exercise in preventing TW‐induced behavioral impairments. Beneficial role of treadmill exercise has been reported in other models of stress. Male Sprague Dawley rats were allocated to five different groups: control (C), social defeat (SD), exercise (Ex), trauma witness (TW), and trauma witness with exercise (TWEx). The two exercised groups, Ex and TWEx, were subjected to a daily 30‐minute treadmill exercise regimen for 14 days. On day 15, the SD group was exposed to 7 days of direct trauma. The trauma witness groups, TW and TWEx, were strategically placed around the cage to witness the SD rats receive social defeat. On day 22, all groups were subjected to behavioral and cognitive tests including short‐term (STM) and long‐term (LTM) memory function, anxiety and depression‐like behaviors. TW and SD had the highest levels of anxiety and depression‐like behaviors compared to the other groups. In addition, TWEx had lower levels of anxiety and depression‐like behaviors compared to TW. STM and LTM remained unchanged. This study demonstrates that treadmill exercise provides resilience to trauma witness in a rodent model.

Support or Funding Information

Grant Support: 2R15MH093918‐02
Investigating the role of the locus coeruleus‐noradrenergic system during social stress on neuroinflammation and resulting changes in serotonin
Authors: Moffitt Casey M , Finnell Julie E , Lombard Calliandra M , Wood Christopher S , Wood Susan K
Published: November 2023

Repeated exposure to social stress is known to result in the emergence of depression. While depression is associated with dysregulation of norepinephrine (NE) and serotonin (5‐HT), the mechanism by which this occurs remains unknown. We have previously determined that a history of social stress enhances neuroinflammation in stress sensitive brain regions, proving causal to a depressive‐like phenotype in socially stressed rats. A growing body of evidence suggests that NE is capable of altering proinflammatory cytokine release (ie. IL‐1β) in the brain. Therefore, there were three major goals of this study: 1) to determine if a history of social defeat produced a sensitized neuroinflammatory response to a subsequent stressor within the serotonergic dorsal raphe (DR); 2) because IL‐1β is capable of decreasing 5‐HT synthesis, we sought to determine whether changes in IL‐β in the DR were related to changes in 5‐HT synthesis and 3) rats were treated with DSP‐4 (400μg/rat, icv), a selective retrograde NE neurotoxin that reduces NE levels in LC target regions (ie., DR), to determine the role of NE on stress‐induced IL‐1β levels in the DR and relative 5‐HT expression. Our initial studies identified sensitized stress‐induced proinflammatory cytokine expression in the brains of socially defeated rats compared with rats with a history of control. Furthermore, DSP‐4 significantly increased stress‐induced proinflammatory cytokines in the central amygdala a prominent LC target and induced anhedonia in the 2‐bottle choice sucrose preference test, regardless of stress history. This suggests that LC‐NE has inhibitory control over neuroinflammation in its target regions, including the DR. Since inflammation is known to increase the enzymatic activity of indoleamine‐2,3‐dioxygenase (IDO), resulting in a shift from 5‐HT synthesis to the production of kynurenine (Kyn), stress‐induced inflammation in the DR is likely associated with a concomitant increase in IDO activity and a reduction of 5‐HT. Together these data suggest that LC‐NE activity may suppress neuroinflammatory drive under conditions of stress and that impaired LC‐NE firing may serve as a mechanistic link between stress‐induced neuroinflammation, dysfunction in the 5‐HT system, and expression of depressive‐like behaviors. These studies seek to identify mechanisms involved in stress susceptibility, in order to reveal novel targets useful in the treatment of stress‐related psychosocial disorders.

Support or Funding Information

15SDG22430017 and P20GM103641
Repeated Social Defeat Stress Changes Peripheral Immune Status in Rats: Potential Effects on Basolateral Amygdala Function
Authors: Munshi Soumyabrata , Rosenkranz J. Amiel
Published: November 2023

Chronic psychological stress is one of the most common triggers of clinical depression and anxiety, and many studies suggest involvement of the immune system in these effects. The basolateral amygdala (BLA), a key brain region involved in emotion, mediates many effects of stress on emotion and may be an intermediary in the link between immune function and depression. Repeated social defeat stress (RSDS) is a powerful model of social stress with known impact on immune function. The peripheral immune system includes pro‐inflammatory and anti‐inflammatory aspects that, as a whole, may exert very different influences on behavior. The aim of this study is (1) to determine the effect of RSDS on the balance between pro‐inflammatory and anti‐inflammatory aspects of peripheral immune function, and (2) to determine if a shift in this balance causes a change in BLA function. Adult male Sprague Dawley rats were exposed to the resident‐intruder model of RSDS using unfamiliar aggressor Long Evans rats for five consecutive days, or control handling. After three days, the following were evaluated: (a) peripheral circulating T‐cell counts and their intracellular cytokine profile (Th1, Th2) by flow‐cytometry, (b) pro‐ and anti‐inflammatory serum cytokine levels by ELISA, and (c) anxiety‐like behavior in the open field test (OFT). RSDS decreased the frequency of dual positive (DP, CD4+CD8+) T‐cells as well as the CD4+ T‐cells with no significant changes in that of the CD8+ T‐cells. Additionally, RSDS increased the frequency of dual negative (DN, CD4‐CD8‐) precursor T‐cells in the circulation. Frequency of CD4+ T‐cells positive for the Th2‐like (anti‐inflammatory) cytokine was increased with similar increase in those of the CD8+ T‐cells positive for the Tc2‐like (anti‐inflammatory) cytokine. RSDS also caused changes in many different serum cytokines. OFT showed that RSDS induced an increase in anxiety‐like behavior. To test whether a pro‐inflammatory state can induce a change in the BLA physiology, in vivo extracellular electrophysiological recordings were performed in a different set of rats that underwent peripheral immune challenge with interleukin‐1β. Interleukin‐1β caused a trend of a time‐dependent alteration of the evoked local field potential as well as alteration of the spontaneous neuronal firing‐rate in the BLA. Taken together, these data suggest that RSDS affects differentiation of peripheral circulatory T‐cells and shifts peripheral immune balance. In addition, a shift in immune balance impacts BLA neuronal activity. This could contribute to the effects of RSDS on anxiety behavior. The results have implications in understanding how chronic stress might alter BLA physiology by recruitment of the peripheral immune system.

Support or Funding Information

NIH Grant MH084970 and MH109484 to JAR.
Caveolin‐2: Improves Longevity and Allows for Stress Adaptation in C. Elegans
Authors: Mehta Meghna D. , Schilling Jan M. , Mehta Ekta D. , Fridolfsson Heidi N. , Roth David M. , Patel Hemal H.
Published: November 2023

Introduction

Caveolins are scaffolding proteins that are involved in stress adaptation via regulation of signal transduction and endocytosis. Three isoforms of caveolin (Cav‐1, Cav‐2, and Cav‐3) are expressed in mammalian cells, while C. elegans express only two (Cav‐1 and Cav‐2). Cav‐2 has conserved homology to the mammalian isoforms Cav‐1 and Cav‐3, with the amino acid region being 67% similar and 37% identical. As aging and diabetic studies are lengthy in mice, a model species such as C. elegans could qualify for high throughput evaluation of the role of caveolin in stress and adaptation since their lifespan is generally under 30 days. Benefits of C. elegans as a model organism are that their whole genome has been sequenced and that they are the only organism to have a complete connectome.

Hypothesis

We hypothesized that expression level of Cav‐2 in C. elegans is a major regulator of stress adaptation with increased expression leading to better, and decreased expression leading to worse stress response.

Methods

We generated C. elegans overexpressing Cav‐2 (Cav‐2 OE) gene through microinjection and transformation with a global promotor. The worms were grown on 1.7% agar plates containing E. coli OP50. We assessed the impact of the Cav‐2 gene on longevity, egg laying, and stress adaptation by comparing the N2 control with the Cav‐2 OE and Cav‐2 KO.

Results

Cav‐2 OE worms showed a 13.3% increase in median lifespan compared to N2 controls and Cav‐2 KO worms (p<0.001, n=180, Log‐Rank Test) under normal conditions. Cav‐2 OE worms had a 28% and 41% increase in production of eggs compared to the N2 controls and Cav‐2 KO worms, respectively. Cav‐2 OE worms also had an 18% increase in the duration of egg laying compared to both N2 controls and Cav‐2 KO worms (p<0.0001, n=180, 2‐Way RM ANOVA). In order to examine the ability of C. elegans to adapt to stress, we induced stress by exposing the C. elegans to 2% glucose. Cav‐2 OE worms showed a 8% and 27% increase in lifespan compared to N2 controls and Cav‐2 KO worms, respectively (p<0.0001, n=180, Log‐Rank Test).

Conclusion

Taken together, our data suggests that Cav‐2 is a major regulator of longevity and stress adaptation in C. elegans and C. elegans may serve as an ideal model system to evaluate the impact of caveolin on human physiology and aging.

Support or Funding Information

This research was supported by funds from the UCSD Department of Anesthesiology.
Anti‐angiogenic Peptide Derived from Kinesin KIF13B Inhibits Trafficking of VEGFR2 to Endothelial Cell surface
Authors: Yamada Kaori , Malik Asrar B.
Published: November 2023

Trafficking of VEGFR2 to the endothelial cell (EC) surface is required for VEGF‐induced angiogenesis. We found that the kinesin‐family plus‐end motor KIF13B transports VEGFR2 to the EC surface. VEGF induced phosphorylation on KIF13B, and activated KIF13B by increasing binding of KIF13B to VEGFR2, and KIF13B‐VEGFR2 complex to microtubule tracks. KIF13B depletion shunted VEGFR2 towards the lysosomal degradation pathway. KIF13B depletion also prevented VEGF‐mediated endothelial migration, capillary tube formation, and neo‐vascularization in mice, suggesting blockade of trafficking as a potential anti‐angiogenesis strategy.

We developed a bioactive peptide based on VEGFR2‐binding domain of KIF13B, which competes specifically with VEGFR2‐binding of KIF13B, and thereby inhibits angiogenesis. The peptide termed a kinesin‐derived angiogenesis inhibitor (KAI) is a cell‐permeable, water‐soluble peptide, inhibiting EC migration, capillary network formation, and EC sprouting, in vitro. KAI also inhibited VEGF‐induced EC migration and tumor growth in human lung carcinoma xenografted in immunodeficient mice. Thus, we describe a novel class of peptides derived from the site of interaction of KIF13B with VEGFR2 that inhibit VEGFR2 trafficking and thereby starve cancer of blood supply.

Support or Funding Information

This work was supported by PPG HL060678, R01 HL045638, P01 HL077806, R01 HL090152, R01 HL118068, and R01 HL125350 to ABM, R56 HL128342 and 13SDG14680053 to KHY.
Mutagenesis study on the Ca2+ sensitivity of the SK2 channel
Authors: Siwiecki Sara Ann , Nam Young Woo , Whitmore Benjamin , Orfali Razan , Zhang Miao
Published: November 2023

The cerebellar Purkinje Cells (PCs) are affected in many types of ataxias. The dysfunction of PCs, more specifically the loss of firing precision of PCs, is one of the primary mechanisms underlying the symptoms of ataxia. Disruptions of regular pacemaking activity of PCs have been identified in studies with mouse models of ataxias. Drugs that normalize the regular firing of PCs have been suggested as therapeutics for the symptom of ataxia patients. Small conductance Ca2+ activated potassium (SK) channels emerged as one of the principle ion channel involved in PCs' pacemaking. Over the past decade, positive allosteric modulators (PAMs) have been developed to target the SK channels. PAMs of SK channels have been shown to alleviate behavioral and neuro‐pathological symptoms in animal models of ataxias. In the SK2 channel, an intrinsically disordered fragment (IDF) at the proximal intracellular C‐terminus connects the transmembrane S6 domain and the CaM binding domain (CaMBD). The flexibility of the IDF makes it a perfect region for the modulation of SK channel activity. The PAMs such as NS309 stabilizes the IDF, and consequently facilitates the coupling between Ca2+ binding to CaM and opening of the channel. This observation motivated us to introduce mutations in the PAM binding pocket, which also result in changes in the Ca2+ sensitivity of SK channels.

Support or Funding Information

American Heart Association SDG Grant, National Ataxia Foundation YI‐SCA Grant to M.Z.
Signaling Pathway by which β‐adrenoceptor Agonists Enhance Vascular Endothelial Growth Factor Release in Activated Human Macrophages
Authors: Ezeamuzie Charles I. , El‐Zohairy Sara , Oriowo Mabayoje
Published: November 2023

Beta‐adrenoceptor agonists such as salbutamol are valuable bronchodilators, but they can also enhance the release of vascular endothelial growth factor (VEGF), which is capable of inducing tissue remodeling and support inflammation. In this study, we have characterized the signaling pathway involved in the enhancement of VEGF release by β‐adrenoceptor agonists in human macrophages. Human U937 cells differentiated into macrophages were primed with LPS in the absence or presence of β‐adrenoceptor agonists and antagonists. The VEGF released and the intracellular cAMP generated were assayed by ELISA. Isoprenaline, procaterol and salbutamol concentration‐dependently enhanced the release of VEGF induced by LPS in U937 macrophages. They also increased intracellular cAMP levels in these cells. BRL 37344, a selective β3‐adrenoceptor agonist, did not enhance VEGF release. Propranolol, ICI 118551 and atenolol all produced a parallel rightward shift of the isoprenaline dose‐response curve. The −logKB values were 8.12 ± 0.17, 8.03 ± 0.05 and 7.23 ± 0.05, respective, respectively, indicating clear involvement of both β1‐ and β2‐adrenoceptor subtypes. Activators of PKA, but not Epac system, enhanced VEGF release, and this effect was abolished by KT 5720 and Rp‐cAMPS ‐ both selective PKA inhibitors. These results show that β1‐ and β2‐, but not β3‐adrenoceptors, mediate cAMP‐dependent enhancement of VEGF release in LPS‐primed differentiated human U937 macrophages, and that PKA, not Epac, was the downstream effector of cAMP activity.

Support or Funding Information

This study was funded by Kuwait University Research Sector Grant # YM 14/09.
Investigating a Novel GPCR ITIM Motif in Regulating Shp2 Binding, Signaling, and Migration
Authors: Paulukinas Ryan D. , Belcastro Lili T. , Moore Catherine C.
Published: November 2023

Aberrant expression of CXCR4, a chemokine G protein‐coupled receptor (GPCR), promotes a migratory and invasive phenotype during breast cancer metastasis, however the mechanism is unknown. Here we investigate the role of CXCR4 tyrosine phosphorylation within a novel ITIM motif. Following activation, CXCR4 is normally phosphorylated on Ser/Thr residues, which promotes receptor desensitization and internalization. We found previously that Ser/Thr phosphorylation of CXCR4 is delayed upon SDF gradient sensing, and correlates with sustained signaling to SHP2, a protein that drives hyperproliferation and invasion of breast cancer. Since SHP2 is known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine‐based inhibitory consensus motifs), we investigated if CXCR4 contains an ITIM motif, and if tyrosine phosphorylation within this motif regulates SHP2 binding, signaling, and migration. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated as measured by recombinant phosphatase assay, phospho‐Ab development, and tyrosine mutagenesis (YF); 2) if tyrosine mutagenesis alters CXCR4 membrane localization, internalization, and SDF gradient sensing, by ELISA; 3) if tyrosine mutagenesis disrupts CXCR4 interaction with SHP2, by co‐IP; 4) if tyrosine mutagenesis alters CXCR4 sustained signaling to SHP2, by Western; and 5) if tyrosine mutation (YF) within the novel ITIM motif in CXCR4 alters migration of metastatic breast cancer cells, by transwell motility assays. Our results demonstrate that CXCR4 phosphorylation is sensitive to recombinant tyrosine phosphatase treatment and tyrosine mutagenesis, suggesting that CXCR4 is indeed tyrosine phosphorylated within the ITIM motif. We determined that while tyrosine phosphorylation within the ITIM motif is not required for CXCR4 membrane localization, internalization, or SDF gradient sensing, it does regulate CXCR4 binding and signaling to SHP2. Investigating the role in CXCR4 mediated migration is on going. To date, our data support a working model that tyrosine phosphorylation of CXCR4 within an ITIM motif is critical for binding to SHP2 and transducing sustained signaling to SHP2. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2.

Support or Funding Information

These studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.
(−)‐Epicatechin Stimulates Mitochondrial Biogenesis in C2C12 Myotubes: Involvement of the G‐protein Coupled Estrogen Receptor (GPER)
Authors: Moreno‐Ulloa Aldo , Licea‐Navarro Alexei , Miranda‐Cervantes Adriana , Beltran‐Partida Ernesto , Donis‐Maturano Luis , De León‐Nava Marco , Villarreal Francisco , Álvarez‐Delgado Carolina
Published: November 2023

During the last decade, it has been of great interest to identify natural products with promising effects on skeletal muscle function. In part, this has been fueled by the interest of developing small molecules with the capability to mimic and/or enhance the effects of exercise in order to improve muscle performance and more importantly to mitigate the effects of aging and disease‐induced muscle wasting. In this regard, our group has provided amply evidence as to how a natural small molecule that belongs to the family of flavonoids (subclass of flavanols), (−)‐epicatechin ((−)‐EPI), is capable to augment exercise capacity in mice and humans associated to an increase in mitochondrial function and biogenesis. The aim of this work was to evaluate the role that the GPER has on mediating the effects of (−)‐EPI on mitochondrial biogenesis in skeletal muscle cells. As an in vitro model of skeletal muscle cells we employed C2C12 myotubes. Using confocal microscopy we demonstrated that the GPER is localized in the cell surface and cytoplasm in C2C12 myotubes. The binding of (−)‐EPI to GPER was evaluated using flow cytometry by means of displacement of estradiol conjugated to fluorescein‐bovine serum albumin (E2‐BSA‐FITC) with increasing concentrations of (−)‐EPI and compared to the potent and selective GPER agonist, G1. To evaluate mitochondrial biogenesis we used fluorescence microscopy in order to quantify mitochondria density accompanied by measurement of oxidative phosphorylation complex proteins II and IV densities by using an in‐cell ELISA kit. Results indicate that (−)‐EPI is capable of displacing E2‐BSA‐FITC similarly to G1. In addition, 48 h of (−)‐EPI treatment (10 M) significantly increases mitochondrial biogenesis in C2C12 myotubes with similar efficacy to that observed with G1, effects that were partially and completely blocked by G36 (GPER antagonist), respectively. Altogether, these results indicate that GPER is expressed in muscle cells and appears to mediate to a significant extent the stimulatory effects of (−)‐EPI on mitochondrial biogenesis. Thus, GPER activation may account for the stimulatory effects on skeletal muscle structure/function observed by either cocoa or pure (−)‐EPI treatment in animals and humans.

Support or Funding Information

NIH DK98717, CONACyT‐CICESE