Drug addiction is characterized by orchestrated transcriptional changes in brain reward regions, including the nucleus accumbens (NAc). The transcription factor E2F3a has emerged as a novel regulator of cocaine’s rewarding effects, yet its sex- and cell-specific mechanisms, as well as its genome-wide targets, remain undetermined. Here, we investigated the motivational and reinforcing roles of E2F3a in cocaine reward using conditioned place preference (CPP) and self-administration, combined with behavioral economics and viral-mediated gene manipulation. Selective overexpression of E2F3a in D1-type medium spiny neurons (MSNs), but not D2-MSNs, increased cocaine CPP in both male and female mice, whereas knockdown produced the opposite effects. Behavioral economics analyses further revealed that E2F3a regulates specific aspects of cocaine reinforcement. Genome-wide mapping revealed increased E2F3a binding to DNA at genes associated with cocaine exposure. Together, these results establish E2F3a as a central substrate of cocaine reward via the recruitment of D1-MSNs and coordinated expression of both proven and new molecular drivers.

Abstract Relapse following prolonged abstinence is a primary challenge in the treatment of opioid and cocaine use disorders, driven in part by enduring dysfunction of medial prefrontal cortex (mPFC) circuits that impair inhibitory control over drug-seeking. The molecular substrates underlying this dysfunction, and whether they differ across drug classes, remain unknown. Here, we performed label-free quantitative proteomics of mPFC synaptosomes isolated from rats after 30-day abstinence following intravenous heroin or cocaine self-administration to profile synaptic adaptations that may contribute to relapse vulnerability. Heroin abstinence induced extensive synaptic proteomic remodeling characterized by coordinated downregulation of mitochondrial proteins involved in oxidative phosphorylation, including pyruvate dehydrogenase complex subunits that regulate carbon entry into mitochondrial metabolism. Targeted metabolomic profiling of whole mPFC revealed accumulation of upstream glycolytic and pentose phosphate pathway intermediates, consistent with altered pyruvate utilization and mitochondrial oxidation. Several bioenergetic metabolites also correlated positively with the severity of escalation of heroin intake. Consistent with the bioenergetic remodeling observed during protracted heroin abstinence, whole-cell patch-clamp recordings from layer V mPFC pyramidal neurons revealed lasting suppression of intrinsic excitability and a decreased spontaneous excitatory synaptic activity. Cocaine abstinence, by contrast, produced limited changes in synaptic bioenergetics while inducing a distinct cytoskeletal remodeling signature. Overall, these findings identify synaptic bioenergetic remodeling as a previously underappreciated feature of prolonged heroin abstinence and reveal a marked divergence in the molecular adaptations induced by heroin versus cocaine within the mPFC. These results implicate mitochondrial bioenergetic pathways as therapeutic targets for reducing relapse vulnerability specifically associated with opioid use disorder.

Although chronic stress increases the risk for depression, only a subset of exposed individuals develop psychiatric illness. The biological mechanisms that protect against depression remain incompletely understood, particularly at the molecular level. Here, we identify a transcriptional network in the nucleus accumbens (NAc), a central brain reward region, that supports stress resilience in both sexes and demonstrate the causal contribution of key hub genes. Using chronic social defeat stress, RNA-seq, and co-expression network analysis, we find sex-specific but overlapping gene modules linked to resilience, anchored by shared hub genes embedded within a common network architecture. Overexpression of these hub genes in stress-naive mice confers stress protection and induces a transcriptional state that is discrete from both susceptible and resilient profiles. These findings position resilience as a structured and targetable molecular phenotype and provide a basis for investigating sex-informed mechanisms of stress adaptation.

Genomic therapy has emerged as a transformative strategy for the prevention, diagnosis and treatment of a wide array of diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis and other CNS-related diseases. Recent developments in chemical strategies and delivery platforms have enhanced the potential of genomic therapies for brain disorders. In this Review, we summarize such strategies, focusing on advances in delivery platforms such as lipid nanoparticles, polymers and oligonucleotide conjugates to facilitate the brain delivery of DNA-based or RNA-based therapeutics into the CNS. We present an overview of the chemical structures and functional moieties of lipids, polymers and oligonucleotides used in these platforms. Lastly, we provide an outlook on future chemical directions to further improve the delivery of genomic medicines to the brain. Genomic therapy offers a promising strategy for addressing central nervous system disorders. This Review highlights recent advances in chemical strategies and delivery platforms, such as lipid nanoparticles, polymers and oligonucleotide conjugates, and it discusses future directions to improve the application of genomic therapy in brain disorders.

Lipid nanoparticle-messenger RNA formulations have garnered significant attention for their therapeutic potential in infectious diseases, cancer and genetic disorders. However, effective mRNA delivery to the central nervous system (CNS) remains a formidable challenge. To overcome this limitation, a class of brain-targeting lipids (BLs) is developed by incorporating brain-targeting small molecules with amino lipids and formulated them with helper lipids to generate brain-targeting lipid nanoparticles (BLNPs) for mRNA delivery. Screening studies led to a lead formulation, TD5 BLNPs, outperforming FDA-approved DLin-MC3-DMA LNPs in delivering mRNA to the brain upon intrathecal injection. Specifically, a single intrathecal injection of TD5 BLNP-GFP mRNA led to GFP expression in 29.6% of neurons and 38.1% of astrocytes across the brain. In an Ai14 mouse model, TD5 BLNP-Cre recombinase mRNA treatment induced tdTomato expression in ≈30% of neurons and 40% of astrocytes across major brain regions. Notably, delivery of Cas9 mRNA/sgRNA complex using TD5 BLNPs achieved effective genome editing in the brain. Additionally, TD5 BLNPs showed comparable safety profiles to MC3 LNPs, indicating promising biocompatibility. Overall, this TD5 BLNP formulation effectively delivers mRNA to brain tissues via intrathecal injection and facilitates efficient expression in both neurons and astrocytes, presenting a potential strategy for treating CNS diseases.

Drug addiction is characterized by neuronal adaptations that support a shift from goal-directed behaviors to habitual, compulsive drug-seeking with persistent effects on cognition and decision-making. Emerging evidence increasingly indicates that astrocytes are also involved in nervous system disorders, including addiction, but the cocaine-induced astrocyte-specific transcriptome has not yet been investigated. We utilized whole cell sorting of astrocytes, RNA-sequencing, and bioinformatic approaches to characterize the astrocyte transcriptome in the nucleus accumbens (NAc), a key brain region involved in reward-processing, following cocaine self-administration, prolonged abstinence, and "relapse" in male mice. We found that astrocytes exhibit robust and contextually-specific transcriptional signatures that converge strongly with human cocaine use disorder. Bioinformatic analysis revealed CREB as a highly ranked predicted upstream regulator of cocaine-induced transcriptional regulation in NAc astrocytes, and CUT&RUN-sequencing mapped increased CREB binding across the astrocyte genome in response to cocaine. Viral-mediated manipulation of CREB activity selectively in NAc astrocytes, in combination with several measures of addiction-related behaviors including conditioned place preference and self-administration, revealed that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. This effect is sex-specific, with no change in astrocytic CREB activity or CPP found in females. Subsequent experiments identify potential molecular mechanisms of astrocytic CREB's influence through modulating astrocytic Ca2+ signaling in response to cocaine. Finally, we show that astrocytic CREB selectively modulates D1-type medium spiny neurons in NAc to control cocaine-related behaviors. Together, these data demonstrate that the astrocyte transcriptome responds robustly to cocaine and that CREB mediates cocaine's effects on gene expression in astrocytes, with consequent effects on neuronal activity and rewarding responses to the drug.

The systemic delivery of mRNA molecules to the central nervous system is challenging as they need to cross the blood-brain barrier (BBB) to reach into the brain. Here we design and synthesize 72 BBB-crossing lipids fabricated by conjugating BBB-crossing modules and amino lipids, and use them to assemble BBB-crossing lipid nanoparticles for mRNA delivery. Screening and structure optimization studies resulted in a lead formulation that has substantially higher mRNA delivery efficiency into the brain than those exhibited by FDA-approved lipid nanoparticles. Studies in distinct mouse models show that these BBB-crossing lipid nanoparticles can transfect neurons and astrocytes of the whole brain after intravenous injections, being well tolerated across several dosage regimens. Moreover, these nanoparticles can deliver mRNA to human brain ex vivo samples. Overall, these BBB-crossing lipid nanoparticles deliver mRNA to neurons and astrocytes in broad brain regions, thereby being a promising platform to treat a range of central nervous system diseases.

Emerging evidence increasingly indicates that astrocytes are involved in neuropsychiatric disorders, including addiction, but the cocaine-induced astrocyte transcriptome has yet to be investigated. We performed RNA sequencing on whole-cell sorted astrocytes from the nucleus accumbens and bioinformatically characterized the astrocyte transcriptome following cocaine self-administration in male mice. We found that astrocytes exhibit robust and contextually specific transcriptional signatures that implicate adenosine 3',5'-monophosphate response element-binding protein (CREB) as a cocaine-induced transcriptional regulator in astrocytes. Cleavage under targets and release using nuclease sequencing (CUT&RUN) revealed increased astrocytic CREB DNA binding in response to cocaine. Viral-mediated manipulation of nucleus accumbens astrocytic CREB activity in combination with addiction-related behaviors revealed that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. Furthermore, we identified potential molecular mechanisms of astrocytic CREB's influence through modulating astrocytic calcium dynamics and selectively increasing D1-type medium spiny neuronal activity.

BACKGROUND: Increasing evidence implicates astrocytes in stress and depression in both rodent models and human major depressive disorder. Despite this, little is known about the transcriptional responses to stress of astrocytes within the nucleus accumbens (NAc), a key brain reward region, and their influence on behavioral outcomes. METHODS: We used whole-cell sorting, RNA sequencing, and bioinformatic analyses to investigate the NAc astrocyte transcriptome in male mice in response to chronic social defeat stress (CSDS). Immunohistochemistry was used to determine stress-induced changes in astrocytic CREB (cAMP response element binding protein) within the NAc. Finally, astrocytic regulation of depression-like behavior was investigated using viral-mediated manipulation of CREB in combination with CSDS. RESULTS: We found a robust transcriptional response in NAc astrocytes to CSDS in stressed mice, with changes seen in both stress-susceptible and stress-resilient animals. Bioinformatic analysis revealed CREB, a transcription factor widely studied in neurons, as one of the top-predicted upstream regulators of the NAc astrocyte transcriptome, with opposite activation states implicated in resilient versus susceptible mice. This bioinformatic deduction was confirmed at the protein level with immunohistochemistry. Moreover, NAc astrocyte morphological complexity correlated with CREB activation and was reduced selectively in astrocytes of resilient mice. Viral overexpression of CREB selectively in NAc astrocytes promoted susceptibility to chronic stress. CONCLUSIONS: Together, our data demonstrate that the astrocyte transcriptome responds robustly to CSDS and that transcriptional regulation in astrocytes contributes to depressive-like behaviors. A better understanding of transcriptional regulation in astrocytes may reveal unknown molecular mechanisms underlying neuropsychiatric disorders.

Delivery of biomacromolecules to the central nervous system (CNS) remains challenging because of the restrictive nature of the blood-brain barrier (BBB). We developed a BBB-crossing conjugate (BCC) system that facilitates delivery into the CNS through γ-secretase-mediated transcytosis. Intravenous administration of a BCC10-oligonucleotide conjugate demonstrated effective transportation of the oligonucleotide across the BBB and gene silencing in wild-type mice, human brain tissues and an amyotrophic lateral sclerosis mouse model.

The development of drug addiction is characterized by molecular changes in brain reward regions that lead to the transition from recreational to compulsive drug use. These neurobiological processes in brain reward regions, such as the nucleus accumbens (NAc), are orchestrated in large part by transcriptional regulation. Our group recently identified the transcription factor E2F3a as a novel regulator of cocaine's rewarding effects and gene expression regulation in the NAc of male mice. Despite this progress, no information is available about the role of E2F3a in regulating cocaine reward at the sex- and cell-specific levels. Here, we used male and female mice expressing Cre-recombinase in either D1- or D2-type medium spiny neurons (MSNs) combined with viral-mediated gene transfer to bidirectionally control levels of E2F3a in a cell-type-specific manner in the NAc during conditioned place preference (CPP) to cocaine. Our findings show that selective overexpression of E2F3a in D1-MSNs increased cocaine CPP in both male and female mice, whereas opposite effects were observed under knockdown conditions. In contrast, equivalent E2F3a manipulations in D2-MSNs had no significant effects. To further explore the role of E2F3a in sophisticated operant and motivated behaviors, we performed viral manipulations of all NAc neurons in combination with cocaine self-administration and behavioral economics procedures in rats and demonstrated that E2F3a regulates sensitivity aspects of cocaine seeking and taking. These results confirm E2F3a as a central substrate of cocaine reward and demonstrate that this effect is mediated in D1-MSNs, thereby providing increased knowledge of cocaine action at the transcriptional level.

Opioid withdrawal is a liability of chronic opioid use and misuse, impacting people who use prescription or illicit opioids. Hyperactive autonomic output underlies many of the aversive withdrawal symptoms that make it difficult to discontinue chronic opioid use. The locus coeruleus (LC) is an important autonomic centre within the brain with a poorly defined role in opioid withdrawal. We show here that pannexin-1 (Panx1) channels expressed on microglia critically modulate LC activity during opioid withdrawal. Within the LC, we found that spinally projecting tyrosine hydroxylase (TH)-positive neurons (LCspinal) are hyperexcitable during morphine withdrawal, elevating cerebrospinal fluid (CSF) levels of norepinephrine. Pharmacological and chemogenetic silencing of LCspinal neurons or genetic ablation of Panx1 in microglia blunted CSF NE release, reduced LC neuron hyperexcitability, and concomitantly decreased opioid withdrawal behaviours in mice. Using probenecid as an initial lead compound, we designed a compound (EG-2184) with greater potency in blocking Panx1. Treatment with EG-2184 significantly reduced both the physical signs and conditioned place aversion caused by opioid withdrawal in mice, as well as suppressed cue-induced reinstatement of opioid seeking in rats. Together, these findings demonstrate that microglial Panx1 channels modulate LC noradrenergic circuitry during opioid withdrawal and reinstatement. Blocking Panx1 to dampen LC hyperexcitability may therefore provide a therapeutic strategy for alleviating the physical and aversive components of opioid withdrawal. Stopping chronic opioid use can lead to withdrawal. Here, authors show in mice that dampening activity of spinally projecting locus coeruleus neurons, targeting microglia, or blocking pannexin-1 channels alleviates opioid withdrawal.

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.

Cocaine use disorder is a significant public health issue without an effective pharmacological treatment. Successful treatments are hindered in part by an incomplete understanding of the molecular mechanisms that underlie long-lasting maladaptive plasticity and addiction-like behaviors. Here, we leverage a large RNA sequencing dataset to generate gene coexpression networks across six interconnected regions of the brain's reward circuitry from mice that underwent saline or cocaine self-administration. We identify phosphodiesterase 1b ( Pde1b ), a Ca 2+ /calmodulin-dependent enzyme that increases cAMP and cGMP hydrolysis, as a central hub gene within a nucleus accumbens (NAc) gene module that was bioinformatically associated with addiction-like behavior. Chronic cocaine exposure increases Pde1b expression in NAc D2 medium spiny neurons (MSNs) in male but not female mice. Viral-mediated Pde1b overexpression in NAc reduces cocaine self-administration in female rats but increases seeking in both sexes. In female mice, overexpressing Pde1b in D1 MSNs attenuates the locomotor response to cocaine, with the opposite effect in D2 MSNs. Overexpressing Pde1b in D1/D2 MSNs had no effect on the locomotor response to cocaine in male mice. At the electrophysiological level, Pde1b overexpression reduces sEPSC frequency in D1 MSNs and regulates the excitability of NAc MSNs. Lastly, Pde1b overexpression significantly reduced the number of differentially expressed genes (DEGs) in NAc following chronic cocaine, with discordant effects on gene transcription between sexes. Together, we identify novel gene modules across the brain's reward circuitry associated with addiction-like behavior and explore the role of Pde1b in regulating the molecular, cellular, and behavioral responses to cocaine.

Opioid use disorder is a chronic relapsing disorder encompassing misuse, dependence, and addiction to opioid drugs. Long term maintenance of associations between the reinforcing effects of the drug and the cues associated with its intake are a leading cause of relapse. Indeed, exposure to the salient drug-associated cues can lead to drug cravings and drug seeking behavior. The dorsal hippocampus (dHPC) and locus coeruleus (LC) have emerged as important structures for linking the subjective rewarding effects of opioids with environmental cues. However, their role in cue-induced reinstatement of opioid use remains to be further elucidated. In this study, we showed that chemogenetic inhibition of excitatory dHPC neurons during re-exposure to drug-associated cues significantly attenuates cue-induced reinstatement of morphine-seeking behavior. In addition, the same manipulation reduced reinstatement of sucrose-seeking behavior but failed to alter memory recall in the object location task. Finally, intact activity of tyrosine hydroxylase (TH) LC-dHPCTh afferents is necessary to drive cue induced reinstatement of morphine-seeking as inhibition of this pathway blunts cue-induced drug-seeking behavior. Altogether, these studies show an important role of the dHPC and LC-dHPCTh pathway in mediating cue-induced reinstatement of opioid seeking.

BACKGROUND:The transcription factor ΔFOSB, acting in the nucleus accumbens, has been shown to control transcriptional and behavioral responses to opioids and other drugs of abuse. However, circuit-level consequences of ΔFOSB induction on the rest of the brain, which are required for its regulation of complex behavior, remain unknown. METHODS:We used an epigenetic approach in mice to suppress or activate the endogenous Fosb gene and thereby decrease or increase, respectively, levels of ΔFOSB selectively in D1-type medium spiny neurons of the nucleus accumbens and tested whether these modifications affect the organization of functional connectivity (FC) in the brain. We acquired functional magnetic resonance imaging data at rest and in response to a morphine challenge and analyzed both stationary and dynamic FC patterns. RESULTS:The 2 manipulations modified brainwide communication markedly and differently. ΔFOSB down- and upregulation had overlapping effects on prefrontal- and retrosplenial cortex-centered networks, but also generated specific FC signatures for epithalamus (habenula) and dopaminergic/serotonergic centers, respectively. Analysis of dynamic FC patterns showed that increasing ΔFOSB essentially altered responsivity to morphine and uncovered striking modifications of the roles of the epithalamus and amygdala in brain communication, particularly upon ΔFOSB downregulation. CONCLUSIONS:These novel findings illustrate how it is possible to link activity of a transcription factor within a single cell type of an identified brain region to consequent changes in circuit function brainwide by use of functional magnetic resonance imaging, and they pave the way for fundamental advances in bridging the gap between transcriptional and brain connectivity mechanisms underlying opioid addiction.

Decades of research advances have established a central role for endogenous opioid systems in regulating reward processing, mood, motivation, learning and memory, gastrointestinal function, and pain relief. Endogenous opioid systems are present ubiquitously throughout the central and peripheral nervous system. They are composed of four families, namely the μ (MOPR), κ (KOPR), δ (DOPR), and nociceptin/orphanin FQ (NOPR) opioid receptors systems. These receptors signal through the action of their endogenous opioid peptides β-endorphins, dynorphins, enkephalins, and nociceptins, respectfully, to maintain homeostasis under normal physiological states. Due to their prominent role in pain regulation, exogenous opioids—primarily targeting the MOPR, have been historically used in medicine as analgesics, but their ability to produce euphoric effects also present high risks for abuse. The ability of pain and opioid use to perturb endogenous opioid system function, particularly within the central nervous system, may increase the likelihood of developing opioid use disorder (OUD). Today, the opioid crisis represents a major social, economic, and public health concern. In this review, we summarize the current state of the literature on the function, expression, pharmacology, and regulation of endogenous opioid systems in pain. Additionally, we discuss the adaptations in the endogenous opioid systems upon use of exogenous opioids which contribute to the development of OUD. Finally, we describe the intricate relationship between pain, endogenous opioid systems, and the proclivity for opioid misuse, as well as potential advances in generating safer and more efficient pain therapies.