Sex-Specific Regulation of Stress Susceptibility by the Astrocytic Gene Htra1.

Major depressive disorder (MDD) is linked to impaired structural and synaptic plasticity in limbic brain regions. Astrocytes, which regulate synapses and are influenced by chronic stress, likely contribute to these changes. We analyzed astrocyte gene profiles in the nucleus accumbens (NAc) of humans with MDD and mice exposed to chronic stress. Htra1 , which encodes an astrocyte-secreted protease targeting the extracellular matrix (ECM), was significantly downregulated in the NAc of males but upregulated in females in both species. Manipulating Htra1 in mouse NAc astrocytes bidirectionally controlled stress susceptibility in a sex-specific manner. Such Htra1 manipulations also altered neuronal signaling and ECM structural integrity in NAc. These findings highlight astroglia and the brain's ECM as key mediators of sex-specific stress vulnerability, offering new approaches for MDD therapies.

A zinc finger transcription factor enables social behaviors while controlling transposable elements and immune response in prefrontal cortex.

The neurobiological origins of social behaviors are incompletely understood. Here we utilized synthetic biology approaches to reprogram the function of ZFP189, a transcription factor whose expression and function in rodent prefrontal cortex was previously demonstrated to be protective against stress-induced social deficits. We created novel synthetic ZFP189 transcription factors including ZFP189VPR, which activates the transcription of target genes and therefore exerts opposite functional control from the endogenous, transcriptionally repressive ZFP189WT. Following viral delivery of these synthetic ZFP189 transcription factors to mouse prefrontal cortex, we observe that ZFP189-mediated transcriptional control promotes mature dendritic spine morphology on transduced pyramidal neurons. Interestingly, inversion of ZFP189-mediated transcription in this brain area, achieved by viral delivery of synthetic ZFP189VPR, precipitates social behavioral deficits in terms of social interaction, motivation, and the cognition necessary for the maintenance of social hierarchy, without other observable behavioral deficits. RNA sequencing of virally manipulated prefrontal cortex tissues reveals that ZFP189 transcription factors of opposing regulatory function (ZFP189WT versus ZFP189VPR) have opposite influence on the expression of genetic transposable elements as well as genes that participate in adaptive immune functions. Collectively, this work reveals that ZFP189 function in the prefrontal cortex coordinates structural and transcriptional neuroadaptations necessary for complex social behaviors while regulating transposable element-rich regions of DNA and the expression of immune-related genes. Given the evidence for a co-evolution of social behavior and the brain immune response, we posit that ZFP189 may have evolved to augment brain transposon-associated immune function as a way of enhancing an animal's capacity for functioning in social groups.

The impact of COVID-19 on mental health and well-being in critical care nurses - a longitudinal, qualitative study.

BACKGROUND: The COVID-19 pandemic has had both a psychological and physiological effect on the human race. For those working in health care, particularly in critical care, the pandemic has put unprecedented strain on staff. Witnessing suffering during crisis in an organizational setting can be a traumatic experience and critical care nurses often risked, not only their own lives, but their psychological well-being, so that those infected with the virus might have a better chance at survival. AIM: The aim of this study was to explore the challenges to mental health and psychological well-being experienced by Critical Care Nurses during the COVID-19 pandemic. STUDY DESIGN: A longitudinal, qualitative study involving semi-structured interviews with 54 critical care nurses across 38 hospitals in the United Kingdom and Ireland. Interviews were transcribed verbatim and analysed using thematic analysis. RESULTS: Four key themes were identified which represent the challenges faces by critical care nurses during the COVID-19 pandemic: Lack of control, Psychological trauma, Unexpected leadership, Public-political betrayal. CONCLUSIONS: While public-political praise may lead to a short-term lift in morale for front line workers; where it is not accompanied by practical support in terms of appropriate equipment, leadership, emotional support and renumeration it is likely to be damaging in the longer term. RELEVANCE TO CLINICAL PRACTICE: This study has provided a greater understanding of the factors which affected the well-being and mental health of critical care nurses during a global pandemic.

What can the uptake of online pharmacy education in Scotland, during the COVID-19 pandemic, tells us about the future of CPD?

Pharmacy CPD in Scotland has been evolving towards online, asynchronous delivery since 2014. The COVID-19 pandemic accelerated this movement by restricting face-to-face education (Zlotos & Stewart, 2021). This study utilised Google analytics to describe web traffic, and electronic e-learning completion records to describe learner activity on the Virtual Learning Environment of a national CPD provider in Scotland. The aim was to describe patterns of learning activity in the years spanning the COVID-19 pandemic, to help predict what future education practice may look like. This study identified that there was an increase in estimated time spent on learning from 8085.5 vs 16,061.5 hours of learning in 2018-19 and 2020-21, respectively. Completion of non-mandatory clinical modules and mandatory service modules increased each year. Mandatory, service focussed modules were most popular each year and the number of completions peaked to coincide with new services or updated content. The findings suggest asynchronous, online pharmacy education continues to grow in popularity. CPD providers should prioritise Mandatory, Service focussed education for pharmacy staff; although, they cannot neglect non-mandatory and Clinical education too. Future education for CPD should be designed to reflect the growing and diverse learner population.

Histone H1x in mouse ventral hippocampus correlates with, but does not cause behavioral adaptations to stress.

Prior research has identified differential protein expression levels of linker histone H1x within the ventral hippocampus (vHipp) of stress-susceptible versus stress-resilient mice. These mice are behaviorally classified based on their divergent responses to chronic social stress. Here, we sought to determine whether elevated vHipp H1x protein levels directly contribute to these diverging behavioral adaptations to stress. First, we demonstrate that stress-susceptible mice uniquely express elevated vHipp H1x protein levels following chronic stress. Given that linker histones coordinate heterochromatin compaction, we hypothesize that elevated levels of H1x in the vHipp may impede pro-resilience transcriptional adaptations and prevent development of the resilient phenotype following social stress. To test this, 8-10-week-old male C57BL/6J mice were randomly assigned to stressed and unstressed groups undergoing 10 days of chronic social defeat stress (CSDS) or single housing respectively. Following CSDS, mice were classified as susceptible versus resilient based on their social interaction behaviors. We synthesized a viral overexpression (OE) vector for H1x and transduced experimental mice with either H1x or control GFP within vHipp. Following viral delivery, we conducted social, anxiety-like, and memory-reliant behavior tests on distinct cohorts of mice. We found no behavioral adaptations following H1x OE compared to GFP controls in susceptible, resilient, or unstressed mice. In sum, although we confirm vHipp protein levels of H1x correlate with susceptibility to social stress, we observe no significant behavioral consequence of H1x OE. Thus, we conclude elevated levels of H1x are correlated with, but are not singularly sufficient to drive development of behavioral adaptations to stress.

Comparison of three DREADD agonists acting on Gq-DREADDs in the ventral tegmental area to alter locomotor activity in tyrosine hydroxylase:Cre male and female rats.

RATIONALE: Despite the increasingly pervasive use of chemogenetic tools in preclinical neuroscience research, the in vivo pharmacology of DREADD agonists remains poorly understood. The pharmacological effects of any ligand acting at receptors, engineered or endogenous, are influenced by numerous factors including potency, time course, and receptor selectivity. Thus, rigorous comparison of the potency and time course of available DREADD ligands may provide an empirical foundation for ligand selection. OBJECTIVES: Compare the behavioral pharmacology of three different DREADD ligands clozapine-N-oxide (CNO), compound 21 (C21), and deschloroclozapine (DCZ) in a locomotor activity assay in tyrosine hydroxylase:cre recombinase (TH:Cre) male and female rats. METHODS: Locomotor activity in nine adult TH:Cre Sprague-Dawley rats (5 female, 4 male) was monitored for two hours following administration of d-amphetamine (vehicle, 0.1-3.2 mg/kg, IP), DCZ (vehicle, 0.32-320 µg/kg, IP), CNO (vehicle, 0.32-10 mg/kg), and C21 (vehicle, 0.1-3.2 mg/kg, IP). Behavioral sessions were conducted twice per week prior to and starting three weeks after bilateral intra-VTA hM3Dq DREADD virus injection. RESULTS: d-Amphetamine significantly increased locomotor activity pre- and post-DREADD virus injection. DCZ, CNO, and C21 did not alter locomotor activity pre-DREADD virus injection. There was no significant effect of DCZ, CNO, and C21 on locomotor activity post-DREADD virus injection; however, large individual differences in both behavioral response and receptor expression were observed. CONCLUSIONS: Large individual variability was observed in both DREADD agonist behavioral effects and receptor expression. These results suggest further basic research would facilitate the utility of these chemogenetic tools for behavioral neuroscience research.

Transcriptional signatures of heroin intake and relapse throughout the brain reward circuitry in male mice.

Opioid use disorder (OUD) looms as one of the most severe medical crises facing society. More effective therapeutics will require a deeper understanding of molecular changes supporting drug-taking and relapse. Here, we develop a brain reward circuit-wide atlas of opioid-induced transcriptional regulation by combining RNA sequencing (RNA-seq) and heroin self-administration in male mice modeling multiple OUD-relevant conditions: acute heroin exposure, chronic heroin intake, context-induced drug-seeking following abstinence, and relapse. Bioinformatics analysis of this rich dataset identified numerous patterns of transcriptional regulation, with both region-specific and pan-circuit biological domains affected by heroin. Integration of RNA-seq data with OUD-relevant behavioral outcomes uncovered region-specific molecular changes and biological processes that predispose to OUD vulnerability. Comparisons with human OUD RNA-seq and genome-wide association study data revealed convergent molecular abnormalities and gene candidates with high therapeutic potential. These studies outline molecular reprogramming underlying OUD and provide a foundational resource for future investigations into mechanisms and treatment strategies.

A zinc finger transcription factor tunes social behaviors by controlling transposable elements and immune response in prefrontal cortex.

The neurobiological origins of social behaviors are incompletely understood. Here we utilized synthetic biology approaches to reprogram the function of ZFP189, a transcription factor whose expression and function in the rodent prefrontal cortex was previously determined to be protective against stress-induced social deficits. We created novel synthetic ZFP189 transcription factors including ZFP189VPR, which activates the transcription of target genes and therefore exerts opposite functional control from the endogenous, transcriptionally repressive ZFP189WT. Upon viral delivery of these synthetic ZFP189 transcription factors to mouse prefrontal cortex, we observe that ZFP189-mediated transcriptional control promotes mature dendritic spine morphology on transduced pyramidal neurons. Interestingly, dysregulation of ZFP189-mediated transcription in this brain area, achieved by delivery of synthetic ZFP189VPR, precipitates social behavioral deficits in terms of social interaction, motivation, and the cognition necessary for the maintenance of social hierarchy, without other observable behavioral deficits. By performing RNA sequencing in virally manipulated prefrontal cortex tissues, we discover that ZFP189 transcription factors of opposing regulatory function have opposite influence on the expression of genetic transposable elements as well as genes that participate in immune functions. Collectively, this work reveals that ZFP189 function in the prefrontal cortex coordinates structural and transcriptional neuroadaptations necessary for social behaviors by binding transposable element-rich regions of DNA to regulate immune-related genes. Given the evidence for a co-evolution of social behavior and the brain immune response, we posit that ZFP189 may have evolved to augment brain transposon-associated immune function as a way of enhancing an animal's capacity for functioning in social groups.

Transcriptional control of nucleus accumbens neuronal excitability by retinoid X receptor alpha tunes sensitivity to drug rewards.

The complex nature of the transcriptional networks underlying addictive behaviors suggests intricate cooperation between diverse gene regulation mechanisms that go beyond canonical activity-dependent pathways. Here, we implicate in this process a nuclear receptor transcription factor, retinoid X receptor alpha (RXRα), which we initially identified bioinformatically as associated with addiction-like behaviors. In the nucleus accumbens (NAc) of male and female mice, we show that although its own expression remains unaltered after cocaine exposure, RXRα controls plasticity- and addiction-relevant transcriptional programs in both dopamine receptor D1- and D2-expressing medium spiny neurons, which in turn modulate intrinsic excitability and synaptic activity of these NAc cell types. Behaviorally, bidirectional viral and pharmacological manipulation of RXRα regulates drug reward sensitivity in both non-operant and operant paradigms. Together, this study demonstrates a key role for NAc RXRα in promoting drug addiction and paves the way for future studies of rexinoid signaling in psychiatric disease states.

Transcriptional signatures of heroin intake and seeking throughout the brain reward circuit.

Opioid use disorder (OUD) looms as one of the most severe medical crises currently facing society. More effective therapeutics for OUD requires in-depth understanding of molecular changes supporting drug-taking and relapse. Recent efforts have helped advance these aims, but studies have been limited in number and scope. Here, we develop a brain reward circuit-wide atlas of opioid-induced transcriptional regulation by combining RNA sequencing (RNAseq) and heroin self-administration in male mice modeling multiple OUD-relevant conditions: acute heroin exposure, chronic heroin intake, context-induced drug-seeking following prolonged abstinence, and heroin-primed drug-seeking (i.e., "relapse"). Bioinformatics analysis of this rich dataset identified numerous patterns of molecular changes, transcriptional regulation, brain-region-specific involvement in various aspects of OUD, and both region-specific and pan-circuit biological domains affected by heroin. Integrating RNAseq data with behavioral outcomes using factor analysis to generate an "addiction index" uncovered novel roles for particular brain regions in promoting addiction-relevant behavior, and implicated multi-regional changes in affected genes and biological processes. Comparisons with RNAseq and genome-wide association studies from humans with OUD reveal convergent molecular regulation that are implicated in drug-taking and relapse, and point to novel gene candidates with high therapeutic potential for OUD. These results outline broad molecular reprogramming that may directly promote the development and maintenance of OUD, and provide a foundational resource to the field for future research into OUD mechanisms and treatment strategies.

CREB Binding at the Zfp189 Promoter Within Medium Spiny Neuron Subtypes Differentially Regulates Behavioral and Physiological Adaptations Over the Course of Cocaine Use.

BACKGROUND: Over the course of chronic drug use, brain transcriptional neuroadaptation is thought to contribute to a change in drug use behavior over time. The function of the transcription factor CREB (cAMP response element binding protein) within the nucleus accumbens (NAc) has been well documented in opposing the rewarding properties of many classes of drugs, yet the gene targets through which CREB causally manifests these lasting neuroadaptations remain unknown. Here, we identify zinc finger protein 189 (Zfp189) as a CREB target gene that is transcriptionally responsive to acute and chronic cocaine use within the NAc of mice. METHODS: To investigate the role of the CREB-Zfp189 interaction in cocaine use, we virally delivered modified clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 constructs capable of selectively localizing CREB to the Zfp189 gene promoter in the NAc of mice. RESULTS: We observed that CREB binding to the Zfp189 promoter increased Zfp189 expression and diminished the reinforcing responses to cocaine. Furthermore, we showed that NAc Zfp189 expression increased within D1 medium spiny neurons in response to acute cocaine but increased in both D1- and D2-expressing medium spiny neurons in response to chronic cocaine. CREB-mediated induction of Zfp189 potentiated electrophysiological activity of D1- and D2-expressing medium spiny neurons, recapitulating the known effect of CREB on these neurons. Finally, targeting CREB to the Zfp189 promoter within NAc Drd2-expressing neurons, but not Drd1-expressing neurons, was sufficient to diminish cocaine-conditioned behaviors. CONCLUSIONS: Together, these findings point to the CREB-Zfp189 interaction within the NAc Drd2+ neurons as a molecular signature of chronic cocaine use that is causal in counteracting the reinforcing effects of cocaine.

Review of the utility of routine mortality reviews among deaths on General Internal Medicine wards in a Canadian tertiary care hospital.

BACKGROUND: Hospital morbidity and mortality reviews are common quality assurance activities, intended to uncover latent or unrecognised systemic issues that contribute to preventable adverse events and patient harm. Mortality reviews may be routinely mandated by hospital policy or for accreditation purposes. However, patients under the care of certain specialties, such as general internal medicine (GIM), are affected by a substantial burden of chronic disease, advanced age, frailty or limited life expectancy. Many of their deaths could be viewed as reasonably foreseeable, and unrelated to poor-quality care. METHODS: We sought to determine how frequently postmortem chart reviews for hospitalised GIM patients at our tertiary care centre in Canada would uncover patient safety or quality of care issues that directly led to these patients' deaths. We reviewed the charts of all patients who died while admitted to the GIM admitting service over a 12-month time period between 1 July 2020 and 30 June 2021. RESULTS: We found that in only 2% of cases was a clinical adverse event detected that directly contributed to a poor or unexpected outcome for the patient, and of those cases, more than half were related to unfortunate nosocomial transmission of COVID-19 infection. CONCLUSION: Due to an overall low yield, we discourage routine mortality chart reviews for general medical patients, and instead suggest that organisations focus on strategies to recognise and capture safety incidents that may not necessarily result in death.

Enhancing Endocannabinoid Signaling via ß-Catenin in the Nucleus Accumbens Attenuates PTSD- and Depression-like Behavior of Male Rats.

Inhibition of fatty acid amide hydrolase (FAAH), which increases anandamide levels, has been suggested as a potential treatment for stress-related conditions. We examined whether the stress-preventing effects of the FAAH inhibitor URB597 on behavior are mediated via ß-catenin in the nucleus accumbens (NAc). Male rats were exposed to the shock and reminders model of PTSD and then treated with URB597 (0.4 mg/kg; i.p.). They were tested for anxiety- (freezing, startle response), depression-like behaviors (despair, social preference, anhedonia), and memory function (T-maze, social recognition). We also tested the involvement of the CB1 receptor (CB1r), ß-catenin, and metabotropic glutamate receptor subtype 5 (mGluR5) proteins. URB597 prevented the shock- and reminders-induced increase in anxiety- and depressive-like behaviors, as well as the impaired memory via the CB1r-dependent mechanism. In the NAc, viral-mediated ß-catenin overexpression restored the behavior of rats exposed to stress and normalized the alterations in protein levels in the NAc and the prefrontal cortex. Importantly, when NAc ß-catenin levels were downregulated by viral-mediated gene transfer, the therapeutic-like effects of URB597 were blocked. We suggest a potentially novel mechanism for the therapeutic-like effects of FAAH inhibition that is dependent on ß-catenin activation in the NAc in a PTSD rat model.

Chromatin-mediated alternative splicing regulates cocaine-reward behavior.

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Opioid withdrawal produces sex-specific effects on fentanyl-vs.-food choice and mesolimbic transcription.

BACKGROUND: Opioid withdrawal is a key driver of opioid addiction and an obstacle to recovery. However, withdrawal effects on opioid reinforcement and mesolimbic neuroadaptation are understudied and the role of sex is largely unknown. METHODS: Male (n=13) and female (n=12) rats responded under a fentanyl-vs.-food "choice" procedure during daily 2h sessions. In addition to the daily choice sessions, rats were provided extended access to fentanyl during 12h self-administration sessions. After two weeks of this self-administration regimen, the nucleus accumbens (NAc) and ventral tegmental area (VTA) of a subset of rats were subjected to RNA sequencing. In the remaining rats, a third week of this self-administration regimen was conducted, during which methadone effects on fentanyl-vs.-food choice were determined. RESULTS: Prior to opioid dependence, male and female rats similarly allocated responding between fentanyl and food. Abstinence from extended fentanyl access elicited similar increases in somatic withdrawal signs in both sexes. Despite similar withdrawal signs and extended access fentanyl intake, opioid withdrawal was accompanied by a maladaptive increase in fentanyl choice in males, but not females. Behavioral sex differences corresponded with a greater number of differentially expressed genes in the NAc and VTA of opioid-withdrawn females relative to males. Methadone blocked withdrawal-associated increases in fentanyl choice in males, but failed to further decrease fentanyl choice in females. CONCLUSIONS: These results provide foundational evidence of sex-specific neuroadaptations to opioid withdrawal, which may be relevant to the female-specific resilience to withdrawal-associated increases in opioid choice and aid in the identification of novel therapeutic targets.

Evaluation of Inter-Professional Education (IPE) with medical, nursing and pharmacy students through a simulated IPL Educational Intervention.

INTRODUCTION: Inter-Professional Education (IPE) is becoming an integral part of many professional programmes throughout the United Kingdom, ensuring health professionals are competent to work as part of an inter-professional team upon entry into their profession. IPE has become a fundamental component of curriculum content in health and social care degrees. AIMS: Research aim - to evaluate a simulated IPE intervention. METHODS: A one day IPE intervention, "Evening On-Call" was run involving nursing and medical students and pre-registration pharmacists (student pharmacists in year 5 of training) in an on-call setting. This IPE incorporated manikin and actor patients in a simulated ward. During the intervention, the 3 groups of students are assessed under observation on their clinical, prioritisation and communication skills. Participants perceptions of this intervention were evaluated by completion of a questionnaire to capture their perceptions regarding the experience, the pre-IPL briefing and post-IPL feedback and perceived relevance of this training. Free text sections collected additional comments and a follow-up questionnaire was sent 6 months later. RESULTS: Initial questionnaire feedback was predominantly positive for each professional group. The majority perceived the simulated IPL had given them a greater understanding of other professionals' roles, had enhanced their professional confidence and would help them prioritise workload once qualified. The 6-months follow-up questionnaire supported the initial questionnaire findings. Some responses highlighted that participants believed the simulated IPL had helped them work more effectively with other healthcare professionals, communicate more effectively and better prioritise their workload. There may be some evidence of sustained self-reported effectiveness in teaching certain professional and clinical skills to participants using this type of simulated intervention.

Gene-Targeted, CREB-Mediated Induction of ΔFosB Controls Distinct Downstream Transcriptional Patterns Within D1 and D2 Medium Spiny Neurons.

BACKGROUND: The onset and persistence of addiction phenotypes are, in part, mediated by transcriptional mechanisms in the brain that affect gene expression and, subsequently, neural circuitry. ΔFosB is a transcription factor that accumulates in the nucleus accumbens (NAc)-a brain region responsible for coordinating reward and motivation-after exposure to virtually every known rewarding substance, including cocaine and opioids. ΔFosB has also been shown to directly control gene transcription and behavior downstream of both cocaine and opioid exposure, but with potentially different roles in D1 and D2 medium spiny neurons (MSNs) in NAc. METHODS: To clarify MSN subtype-specific roles for ΔFosB and investigate how these coordinate the actions of distinct classes of addictive drugs in NAc, we developed a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-based epigenome editing tool to induce endogenous ΔFosB expression in vivo in the absence of drug exposure. After inducing ΔFosB in D1- or D2-MSNs or both, we performed RNA sequencing on bulk male and female NAc tissue (n = 6-8/group). RESULTS: We found that ΔFosB induction elicits distinct transcriptional profiles in NAc by MSN subtype and by sex, establishing for the first time that ΔFosB mediates different transcriptional effects in males versus females. We also demonstrated that changes in D1-MSNs, but not those in D2-MSNs or both, significantly recapitulate changes in gene expression induced by cocaine self-administration. CONCLUSIONS: Together, these findings demonstrate the efficacy of a novel molecular tool for studying cell type-specific transcriptional mechanisms and shed new light on the activity of ΔFosB, a critical transcriptional regulator of drug addiction.

A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila Melanogaster.

Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP2 also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward.

Chronic stress and antidepressant treatment alter purine metabolism and beta oxidation within mouse brain and serum.

Major depressive disorder (MDD) is a complex condition with unclear pathophysiology. Molecular disruptions within limbic brain regions and the periphery contribute to depression symptomatology and a more complete understanding the diversity of molecular changes that occur in these tissues may guide the development of more efficacious antidepressant treatments. Here, we utilized a mouse chronic social stress model for the study of MDD and performed metabolomic, lipidomic, and proteomic profiling on serum plus several brain regions (ventral hippocampus, nucleus accumbens, and medial prefrontal cortex) of susceptible, resilient, and unstressed control mice. To identify how commonly used tricyclic antidepressants impact the molecular composition in these tissues, we treated stress-exposed mice with imipramine and repeated our multi-OMIC analyses. Proteomic analysis identified three serum proteins reduced in susceptible animals; lipidomic analysis detected differences in lipid species between resilient and susceptible animals in serum and brain; and metabolomic analysis revealed dysfunction of purine metabolism, beta oxidation, and antioxidants, which were differentially associated with stress susceptibility vs resilience by brain region. Antidepressant treatment ameliorated stress-induced behavioral abnormalities and affected key metabolites within outlined networks, most dramatically in the ventral hippocampus. This work presents a resource for chronic social stress-induced, tissue-specific changes in proteins, lipids, and metabolites and illuminates how molecular dysfunctions contribute to individual differences in stress sensitivity.