Investigation of the Adenosine A(2A) Receptor on the Enhanced Rewarding Effects of Nicotine and Dopamine D2 Receptor Signaling in a Novel Heritable Model of Drug Abuse Vulnerability

Investigation of the adenosine A(2A) receptor on the enhanced rewarding effects of nicotine and dopamine D2 receptor signaling in a novel heritable model of drug abuse vulnerability Seth E. Turney, Loren D. Peeters, Olivia A. Jennings, Liza J. Wills, Russell W. Brown Many years ago, our laboratory along with a collaborator established that neonatal treatment of the dopamine (DA)D2-like receptor agonist quinpirole (NQ) to rats induces an increase in DAD2 receptor sensitivity throughout the animal’s lifetime, which has validity to schizophrenia (SZ) and a number of clinical conditions. These clinical conditions, which include SZ but also bipolar disorder, obsessive-compulsive disorder, panic disorder, and major depression all demonstrate increased drug abuse vulnerability, especially to cigarette smoking. Based on this permanent change in DAD2 sensitivity, we bred NQ-treated male and female rats with their NQ-treated or neonatal saline (NS)-treated counterparts. This F1 generation also demonstrated increases in DAD2 signaling, both behaviorally as well as through DAD2 signaling mechanisms. We have shown both d enhanced behavioral responding to nicotine on the conditioned place preference (CPP) and behavioral sensitization paradigms. These F1 offspring of NQ-treated rats also demonstrated increases of G-protein dependent and G-protein independent DAD2 signaling. Interestingly, the adenosine A(2A) receptor forms a mutual inhibitory heteromer with the DAD2 receptor. Adenosine is a known neuromodulator that can increase or decrease synaptic transmission in the brain, and there exists a hypothesis that adenosine dysfunction is the primary system which is disrupted in SZ which leads to changes in the dopamine and other neurotransmitter systems. The drug CGS 21680, an A(2A) agonist which stimulates the A(2A) receptor, is known to decrease DAD2 signaling and has been shown to block nicotine behavioral sensitization. A major focus in this project is on the adenosine A(2A) receptor as a novel pharmacological treatment target, since it is known that antipsychotic drugs which are often used to treat SZ and these other clinical conditions which have increased DAD2 signaling produce deleterious side effects, and novel medications are needed. Results here revealed that a 0.09 mg/kg dose of CGS 21680 was effective to block enhanced nicotine CPP and changes in DAD2 G-protein independent signaling in F1 generation rats. Interestingly, CGS 21680 did not affect G-protein dependent signaling in F1 generation animals, suggesting that the mechanism through which it is working may not be through the traditional DAD2 signaling mechanism. Future work is designed to analyze underlying mechanisms of this effect

Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received

Background The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective To report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, setting, and participants This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment. Outcome measurements and statistical analysis Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and limitations According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa. Conclusions Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient summary More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common

Functional and quality of life outcomes of localised prostate cancer treatments (prostate testing for cancer and treatment [ProtecT] study)

Objective To investigate the functional and quality of life (QoL) outcomes of treatments for localised prostate cancer and inform treatment decision-making. Patients and Methods Men aged 50–69 years diagnosed with localised prostate cancer by prostate-specific antigen testing and biopsies at nine UK centres in the Prostate Testing for Cancer and Treatment (ProtecT) trial were randomised to, or chose one of, three treatments. Of 2565 participants, 1135 men received active monitoring (AM), 750 a radical prostatectomy (RP), 603 external-beam radiotherapy (EBRT) with concurrent androgen-deprivation therapy (ADT) and 77 low-dose-rate brachytherapy (BT, not a randomised treatment). Patient-reported outcome measures (PROMs) completed annually for 6 years were analysed by initial treatment and censored for subsequent treatments. Mixed effects models were adjusted for baseline characteristics using propensity scores. Results Treatment-received analyses revealed different impacts of treatments over 6 years. Men remaining on AM experienced gradual declines in sexual and urinary function with age (e.g., increases in erectile dysfunction from 35% of men at baseline to 53% at 6 years and nocturia similarly from 20% to 38%). Radical treatment impacts were immediate and continued over 6 years. After RP, 95% of men reported erectile dysfunction persisting for 85% at 6 years, and after EBRT this was reported by 69% and 74%, respectively (P < 0.001 compared with AM). After RP, 36% of men reported urinary leakage requiring at least 1 pad/day, persisting for 20% at 6 years, compared with no change in men receiving EBRT or AM (P < 0.001). Worse bowel function and bother (e.g., bloody stools 6% at 6 years and faecal incontinence 10%) was experienced by men after EBRT than after RP or AM (P < 0.001) with lesser effects after BT. No treatment affected mental or physical QoL. Conclusion Treatment decision-making for localised prostate cancer can be informed by these 6-year functional and QoL outcomes

Behavioral and Neuroinflammatory Sex Differences in Comorbid Posttraumatic Stress Disorder and Alcohol Use Disorder

Post-traumatic stress disorder (PTSD) is a debilitating disorder with a prevalence rate of approximately 5%. Unfortunately, this disorder is commonly associated with another debilitating disorder, alcohol use disorder (AUD). Of the 5% of people diagnosed with PTSD, 30%-59% also suffer from AUD. Currently, there are limited effective treatment options for those suffering from comorbid PTSD/AUD. Previous research has suggested that biological sex differentially impacts PTSD comorbid with AUD, however, the underlying mechanisms are enigmatic. The goal of this study was to better understand the underlying mechanisms that mediate sex differences in a rodent model of comorbid PTSD/AUD by analyzing specific behavioral tasks and changes in neuronal function of specific brain regions. Chronic inflammation has been implicated in PTSD and AUD respectively, with differences between sexes being observed. Females tend to express elevated levels of inflammation in both disorders compared to males in brain regions such as, the hippocampus, amygdala, and prefrontal cortex. Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that is released during neuronal inflammation. To further examine these sex differences, a comorbid PTSD/AUD rodent model was implemented using restraint stress (RS) and chronic intermittent ethanol use (CIE). Following the exposure to RS and CIE a fear conditioning procedure was implemented to assess changes in future stress sensitivity. The fear conditioning paradigm was accomplished by conditioning the animal to pair a tone with a foot shock, followed by extinction of that behavior in a different context where the animal received the tone but no foot shock. Thereafter, the animal was placed back in the context they received the foot shock, known as context renewal, but acquired no tone or foot shock. The behavior in these different contexts was analyzed to test memory and stress sensitivity. Brain tissue was collected to analyze TNF-α protein expression in regions associated with learning, memory, and addiction such as, the prelimbic cortex (PrL), infralimbic cortex (IfL), and the hippocampus. The results of the fear conditioning revealed that the females froze more altogether compared to the males, and there was more freezing of the females with RS and CIE during context renewal. It is expected that TNF-α protein expression will be significantly elevated in females when compared to males, regardless of treatment group. Females exposed to RS and CIE will have significantly higher TNF-α levels when compared to all other treatment groups. Finally, increases in TNF-α protein expression will be region specific with the PrL and IfL regions exhibiting significantly greater expression than the hippocampus. This study will aid in better understanding the sex differences and lead to better treatment options that are sex-dependent for those diagnosed with comorbid PTSD/AUD

CDPPB attenuates risky behavior in a rodent model of PTSD/AUD comorbidity

Alcohol use disorder (AUD) is the leading cause of substance use disorders among Veterans and 55 to 75% of the population that are diagnosed with PTSD also receive a comorbid diagnosis of AUD. The co-diagnosis of PTSD/AUD is associated with neurocognitive changes such as increased impulsivity and risk-taking behavior, especially among individuals with combat-related trauma. Furthermore, increased neuroinflammation in subregions of the prefrontal cortex (PFC) are suggested to contribute to these neurocognitive changes. To better understand the cognitive deficits associated with co-occurring PTSD/AUD we incorporated a probabilistic discounting task (PDT) to model risk-based decision-making in male and female Wistar rats that were exposed to restraint stress (RS) and chronic intermittent ethanol exposure (CIE). Following RS and CIE, rats underwent lever press training through a series of different training phases, in which one lever delivered a small reward 100% of the time, and the other a large reward, delivered with descending probability each trial block. Pressing the large-reward lever during the final two trial blocks when it is disadvantageous to do so is considered “risky” behavior. A week prior to PDT, rats were treated prophylactically with CDPPB, a positive allosteric modulator of the metabotropic glutamate type 5 (mGlu5) receptor, to determine if the cognitive deficits caused by stress and alcohol exposure could be prevented. Additionally, to determine if our model mimicked the neuroinflammatory mechanism seen in the human condition and the therapeutic effects of CDPPB, we assessed TNF-⍺ protein expression in a subset of rats. Our results indicated that male rats exposed to RS and CIE had significantly greater responding during the 3rd, 4th, and 5th risk blocks compared to all other groups. However, the administration of CDPPB reversed this effect. Females exposed to RS and CIE only displayed increased risky behavior at the highest risk block and this was also blocked with the administration of CDPPB. We also determined that RS and CIE significantly increased TNF-⍺ levels in the IfL cortex compared to either RS or CIE alone and the prophylactic administration of CDPPB reduced TNF-⍺ protein expression to control animal levels. In the present study, we demonstrate that exposure to stress and chronic alcohol leads to significant neurocognitive deficits resulting in increased risky decision-making, but these deficits can be attenuated through modulation of the mGlu5 receptor prior to behavioral testing. Additionally, these deficits could be due to deleterious neuroinflammation in subregions of the PFC

Reinstatement of nicotine conditioned place preference in a transgenerational model of drug abuse vulnerability in psychosis: Impact of BDNF on the saliency of drug associations

Rationale: Psychotic disorders such as schizophrenia are often accompanied by high rates of cigarette smoking, reduced quit success, and high relapse rates, negatively affecting patient outcomes. However, the mechanisms underlying altered relapse-like behaviors in individuals diagnosed with psychosis are poorly understood. Objectives: The present study analyzed changes in extinction and reinstatement of nicotine conditioned place preference (CPP) and resulting changes in brain-derived neurotrophic factor (BDNF) in a novel heritable rodent model of psychosis, demonstrating increased dopamine D2 receptor sensitivity, to explore mechanisms contributing to changes in relapse-like behaviors. Methods: Male and female offspring of two neonatal quinpirole-treated (QQ) and two neonatal saline-treated (SS) Sprague-Dawley rats (F1 generation) were tested on an extended CPP paradigm to analyze extinction and nicotine-primed reinstatement. Brain tissue was analyzed 60 min after the last nicotine injection for BDNF response in the ventral tegmental area (VTA), the infralimbic (IfL) and prelimbic (PrL) cortices. Results: F1 generation QQ offspring demonstrated delayed extinction and more robust reinstatement compared to SS control animals. In addition, QQ animals demonstrated an enhanced BDNF response to nicotine in the VTA, IfL and Prl cortices compared to SS offspring. Conclusions: This study is the first to demonstrate altered relapse-like behavior in a heritable rodent model with relevance to comorbid drug abuse and psychosis. This altered pattern of behavior is hypothesized to be related to elevated activity-dependent BDNF in brain areas associated with drug reinforcement during conditioning that persists through the extinction phase, rendering aberrantly salient drug associations resistant to extinction and enhancing relapse vulnerability

Modulation of mGlu5 Improves Sensorimotor Gating Deficits in Rats Neonatally Treated With Quinpirole Through Changes in Dopamine D2 Signaling

This study analyzed whether the positive allosteric modulator of metabotropic glutamate receptor type 5 (mGlu5) 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) would alleviate deficits in prepulse inhibition (PPI) and affect dopamine (DA) D2 signaling in the dorsal striatum and prefrontal cortex (PFC) in the neonatal quinpirole (NQ) model of schizophrenia (SZ). Male and female Sprague-Dawley rats were neonatally treated with either saline (NS) or quinpirole HCL (1 mg/kg; NQ), a DAD2 receptor agonist, from postnatal days (P) 1–21. Rats were raised to P44 and behaviorally tested on PPI from P44-P48. Before each trial, rats were subcutaneous (sc) administered saline or CDPPB (10 mg/kg or 30 mg/kg). On P50, rats were given a spontaneous locomotor activity test after CDPPB or saline administration. On P51, the dorsal striatum and PFC were evaluated for both arrestin-2 (βA-2) and phospho-AKT protein levels. NQ-treated rats demonstrated a significant deficit in PPI, which was alleviated to control levels by the 30 mg/kg dose of CDPPB. There were no significant effects of CDPPB on locomotor activity. NQ treatment increased βA-2 and decreased phospho-AKT in both the dorsal striatum and PFC, consistent with an increase DAD2 signaling. The 30 mg/kg dose of CDPPB significantly reversed changes in βA-2 in the dorsal striatum and PFC and phospho-AKT in the PFC equivalent to controls. Both doses of CDPPB produced a decrease of phospho-AKT in the PFC compared to controls. This study revealed that a mGlu5 positive allosteric modulator was effective to alleviate PPI deficits and striatal DAD2 signaling in the NQ model of SZ

Behavioral Effects and Neurobiological Mechanisms of 3-Aminobenzimide in a Rodent Model of Chronic Psychological Stress

Major depressive disorder (MDD) is a leading cause of disability worldwide, with a lifetime prevalence rate of approximately 20%. Inadequate pharmacological treatment methods for MDD are a significant debilitating factor. Patient estimates suggest that the treatment resistance rate for pharmacological interventions is over 30%. Postmortem analyses of human tissue of individuals diagnosed with MDD have shown an increase in Poly (ADP-ribose) polymerase 1 (PARP-1) mRNA gene expression in prefrontal cortical white matter when compared to psychiatrically normal brain tissue. In order to further investigate this issue, the present study used the social defeat stress/chronic unpredictable stress (SDS + CUS) rodent model of depression to induce a state of chronic psychological distress. Rats were treated with either the PARP-inhibitor, 3- aminobenzamide (3-AB); a common selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX), or saline. During the stress manipulation we conducted the sucrose preference test, results revealed that saline-treated rats which had undergone SDS + CUS showed significant reductions in sucrose preference compared to all other groups. In addition, a social interaction test was conducted one day after the stress manipulation, and saline-treated stressed animals demonstrated less social interaction compared to all other groups, indicating the stress manipulation was effective. Neurobiological assays were conducted to examine PARP expression, microglial morphology, and proinflammatory cytokine expression. Though we expected to find a decrease, results from immunofluorescence studies of tissue sections revealed an elevation of PARP-1 protein expression in prefrontal cortical gray matter in the FLX/Stress group compared with SAL/Stress group. Microglial morphological changes indicated that the SAL/Stress group had significantly more prolate microglia when compared to all other treatment groups, suggesting early activation of microglia, an indicator of neuroinflammation. Increases in IL-1β and TNF-⍺ expression was observed in the hippocampus of the SAL/Stress group when compared to all other treatment groups. Interestingly, IL-6 expression was significantly elevated in the SAL/Stress group when compared to the FLX/Stress group and theCTRL/No stress group but did not significantly differ from the 3-AB/Stress group. This study revealed therapeutic potential of 3-AB for the treatment of stress-related disorders, as well as the neuroinflammatory mechanisms associated with chronic stress

Metabotropic Glutamate Receptor Type 5 (Mglu5) as a Therapeutic Target Towards the Enhanced Rewarding Effects of Nicotine and Deficits in Sensorimotor Gating in a Heritable Model of Drug Abuse Vulnerability in Psychosis

Heritable and environmental factors contribute to an individual’s risk of substance abuse and psychosis. Individuals diagnosed with a mental disorder have greater vulnerability for substance abuse. Our laboratory established that neonatal treatment of rats with quinpirole (NQ), a dopamine (DA) D2-like agonist, results in a significant increase of DAD2 receptor sensitivity throughout the animal’s lifetime. An increase of DAD2 receptor sensitivity is relevant to a model of schizophrenia (SZ), although increases of DAD2 receptor activity also occur in a number of clinical disorders, including bipolar disorder, obsessive-compulsive disorder, panic disorder, and major depression. Common amongst these clinical conditions is a dramatic increase in cigarette smoking compared to the general population. We bred NQ-treated male and female rats with their NQ-treated or neonatal saline (NS)-treated counterparts once they reached adulthood to determine whether increases in DAD2 sensitivity were passed to the next generation. Offspring of these animals, regardless of whether one or both founders received NQ-treatment, also demonstrated increases of DAD2 receptor sensitivity both behaviorally and neurobiologically. RNASeq preliminary data revealed an increase in cortisol synthesis and release in F1 generation animals, demonstrating an enhanced response to stress, consistent with a model of drug abuse vulnerability. Consistent with this finding, F1 generation rats demonstrated enhanced nicotine conditioned place preference (CPP) and had an enhanced brain derived neurotrophic factor (BDNF) response to nicotine in the nucleus accumbens (NAcc), a brain area critical to drug reward. The DAD2 receptor forms a triple heteromer with the adenosine A(2A) and metabotropic glutamate type 5 (mGlu5) receptor, such that stimulation of either receptor results in a decrease of DAD2 activity. Therefore, we analyzed whether use of a positive allosteric modulator (PAM) of mGlu5 in the F1 generation would block nicotine CPP and improve sensorimotor gating deficits, which is a hallmark of psychosis. In both experiments, the mGlu5 PAM effectively blocked the enhanced rewarding effects of nicotine and also alleviated sensorimotor gating deficits in this model. In essence, we demonstrate in results reported here that there may be a common therapeutic target for the dual treatment of substance abuse and psychosis

Modulation of Metabotropic Glutamate Receptor Type 5 (mGlu5) Reduces the Enhanced Rewarding Effects of Nicotine in a Neonatal Quinpirole Model of Psychosis

Nicotine has been indicated as a prevalent drug for substance abuse comorbidities in mental illness. Tobacco use is elevated in those suffering from psychiatric disorders, most notably in schizophrenia (SZ), where a three-to-five fold increase in usage compared to the general population is observed. Our laboratory has established a rodent model of psychosis. In this model, male and female rats are neonatally treated with quinpirole (NQ), a dopamine (DA) D2-like agonist for 21 days postpartum, resulting in lifelong supersensitization of the DAD2 receptor. Increases in dopamine D2 receptor sensitivity is a hallmark of psychosis. Interestingly, the dopamine D2 receptor forms a triple mutual inhibitor heteromer in the dorsal striatum with the adenosine A(2A) and metabotropic glutamate receptor type 5 (mGlu5), such that stimulation of the A(2A) or mGlu5 receptor results in decreased dopamine D2 signaling. The present study was designed to analyze the role of the mGlu5 receptor in a behavioral task involved in testing the associative aspects of rewarding drugs known as conditioned place preference (CPP). CPP is a behavioral task in which animals are conditioned with a reinforcing drug to prefer a particular environmental context. Male and female rats were neonatally treated with saline (NS) or quinpirole from postnatal day (P) 1 to 21. From P41-51, which is mid-adolescence in a rat, all rats were behaviorally tested on CPP. Results revealed that compared to NS rats, NQ animals administered nicotine demonstrated enhanced CPP, replicating our past work. Groups receiving a positive allosteric modulator to mGlu5, which results in stimulation of the mGlu5 receptor, reduced the enhanced rewarding effects of nicotine in CPP for NQ treated rats equal to control levels. Brain tissue was analyzed for brain-derived neurotropic factor (BDNF), a neurotrophin involved in cell growth, as well cell adhesion molecule cadherin-13 in the ventral tegmental area (VTA), which is a brain area rich in dopamine cell bodies. Results revealed elevations of BDNF in NQ-treated rats given nicotine compared to all other groups, and a sex difference in the increase in cadherin-13, with female NQ rats given nicotine demonstrating increases compared to all other groups. These effects were blocked by the mGlu5 receptor positive allosteric modulator. In addition, we analyzed phospho-p70S6 kinase in the nucleus accumbens (NAcc), which is the dopamine neuronal terminal region in the VTA mitigating drug reward. The NQ group given nicotine demonstrated significant increases in NAcc P70S6 kinase compared to all other groups, suggesting increased synaptic growth, which was also blocked by the positive allosteric modulator to mGlu5. Taken together, these results elucidate mGlu5 as a drug target for reducing the rewarding effects of nicotine via CDPPB administration in a model of substance abuse in psychosis