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The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with pain therapeutics. We developed a mouse model of oxycodone exposure and subsequent withdrawal in the presence or absence of chronic neuropathic pain. Oxycodone withdrawal alone triggered robust gene expression adaptations in the nucleus accumbens, medial prefrontal cortex and ventral tegmental area, with numerous genes and pathways selectively affected by oxycodone withdrawal in mice with peripheral nerve injury. Pathway analysis predicted that histone deacetylase (HDAC) 1 is a top upstream regulator in opioid withdrawal in nucleus accumbens and medial prefrontal cortex. The novel HDAC1/HDAC2 inhibitor, Regenacy Brain Class I HDAC Inhibitor (RBC1HI), attenuated behavioral manifestations of oxycodone withdrawal, especially in mice with neuropathic pain. These findings suggest that inhibition of HDAC1/HDAC2 may provide an avenue for patients with chronic pain who are dependent on opioids to transition to non-opioid analgesics.

Opioid analgesics exert their therapeutic and adverse effects by activating μopioid receptors (MOPR); however, functional responses to MOPR activation are modulated by distinct signal transduction complexes within the brain. The ventrolateral periaqueductal gray (vlPAG) plays a critical role in modulation of nociception and analgesia, but the exact intracellular pathways associated with opioid responses in this region are not fully understood. We previously showed that knockout of the signal transduction modulator Regulator of G protein Signaling z1 (RGSz1) enhanced analgesic responses to opioids, whereas it decreased the rewarding efficacy of morphine. Here, we applied viral mediated gene transfer methodology and delivered adeno-associated virus (AAV) expressing Cre recombinase to the vlPAG of RGSz1fl\flmice to demonstrate that downregulation of RGSz1 in this region decreases sensitivity to morphine in the place preference paradigm, under pain-free as well as neuropathic pain states. We also used retrograde viral vectors along with flippase-dependent Cre vectors to conditionally downregulate RGSz1 in vlPAG projections to the ventral tegmental area (VTA) and show that downregulation of RGSz1 prevents the development of place conditioning to low morphine doses. Consistent with the role for RGSz1 as a negative modulator of MOPR activity, RGSz1KO enhances opioid-induced cAMP inhibition in periaqueductal gray (PAG) membranes. Furthermore, using a new generation of bioluminescence resonance energy transfer (BRET) sensors, we demonstrate that RGSz1 modulates Gαz but not other Gαi family subunits and selectively impedes MOPR-mediated Gαz signaling events invoked by morphine and other opioids. Our work highlights a regional and circuit-specific role of the G protein–signaling modulator RGSz1 in morphine reward, providing insights on midbrain intracellular pathways that control addiction-related behaviors.SIGNIFICANCE STATEMENTThis study used advanced genetic mouse models to highlight the role of the signal transduction modulator named RGSz1 in responses to clinically used opioid analgesics. We show that RGSz1 controls the rewarding efficacy of opioids by actions in ventrolateral periaqueductal gray projections to the ventral tegmental area, a key component of the midbrain dopamine pathway. These studies highlight novel mechanisms by which pain-modulating structures control the rewarding efficacy of opioids.

Major depressive disorder (MDD) is the leading cause of disability worldwide. There is an urgent need for objective biomarkers to diagnose this highly heterogeneous syndrome, assign treatment, and evaluate treatment response and prognosis. MicroRNAs (miRNAs) are short non-coding RNAs, which are detected in body fluids that have emerged as potential biomarkers of many disease conditions. The present study explored the potential use of miRNAs as biomarkers for MDD and its treatment. We profiled the expression levels of circulating blood miRNAs from mice that were collected before and after exposure to chronic social defeat stress (CSDS), an extensively validated mouse model used to study depression, as well as after either repeated imipramine or single-dose ketamine treatment. We observed robust differences in blood miRNA signatures between stress-resilient and stress-susceptible mice after an incubation period, but not immediately after exposure to the stress. Furthermore, ketamine treatment was more effective than imipramine at re-establishing baseline miRNA expression levels, but only in mice that responded behaviorally to the drug. We identified the red blood cell-specific miR-144-3p as a candidate biomarker to aid depression diagnosis and predict ketamine treatment response in stress-susceptible mice and MDD patients. Lastly, we demonstrate that systemic knockdown of miR-144-3p, via subcutaneous administration of a specific antagomir, is sufficient to reduce the depression-related phenotype in stress-susceptible mice. RNA-sequencing analysis of blood after such miR-144-3p knockdown revealed a blunted transcriptional stress signature as well. These findings identify miR-144-3p as a novel target for diagnosis of MDD as well as for antidepressant treatment, and enhance our understanding of epigenetic processes associated with depression.

Animals susceptible to chronic social defeat stress (CSDS) exhibit depression-related behaviors, with aberrant transcription across several limbic brain regions, most notably in the nucleus accumbens (NAc). Early life stress (ELS) promotes susceptibility to CSDS in adulthood, but associated enduring changes in transcriptional control mechanisms in the NAc have not yet been investigated. In this study, we examined long-lasting changes to histone modifications in the NAc of male and female mice exposed to ELS. Dimethylation of lysine 79 of histone H3 (H3K79me2) and the enzymes (DOT1L and KDM2B) that control this modification are enriched in D2-type medium spiny neurons and are shown to be crucial for the expression of ELS-induced stress susceptibility. We mapped the site-specific regulation of this histone mark genome wide to reveal the transcriptional networks it modulates. Finally, systemic delivery of a small molecule inhibitor of DOT1L reversed ELS-induced behavioral deficits, indicating the clinical relevance of this epigenetic mechanism.

Low miR-218 expression in the medial prefrontal cortex (mPFC) is a consistent trait of depression. Here we assessed whether miR-218 in the mPFC confers resilience or susceptibility to depression-like behaviors in adult mice, using the chronic social defeat stress (CSDS) model of depression. We also investigated whether stress-induced variations of miR-218 expression in the mPFC can be detected in blood. We find that downregulation of miR-218 in the mPFC increases susceptibility to a single session of social defeat, whereas overexpression of miR-218 selectively in mPFC pyramidal neurons promotes resilience to CSDS and prevents stress-induced morphological alterations to those neurons. After CSDS, susceptible mice have low levels of miR-218 in blood, as compared with control or resilient groups. We show further that upregulation and downregulation of miR-218 levels specifically in the mPFC correlate with miR-218 expression in blood. Our results suggest that miR-218 in the adult mPFC might function as a molecular switch that determines susceptibility vs. resilience to chronic stress, and that stress-induced variations in mPFC levels of miR-218 could be detected in blood. We propose that blood expression of miR-218 might serve as potential readout of vulnerability to stress and as a proxy of mPFC function.

Depression is a devastating psychiatric disorder caused by a combination of geneticpredisposition and life events, mainly exposure to stress. Early life stress (ELS) inparticular is known to “scar” the brain, leading to an increased susceptibility todeveloping depression later in life via epigenetic mechanisms. Epigenetic processeslead to changes in gene expression that are not due to changes in DNA sequence, butachieved via modulation of chromatin modifications, DNA methylation, and noncodingRNAs. Here we review common epigenetic mechanisms including the enzymes that takepart in reading, writing, and erasing specific epigenetic marks. We then describerecent developments in understanding how ELS leads to changes in the epigenome thatare manifested in increased susceptibility to depression-like abnormalities in animalmodels. We conclude with highlighting the need for future studies that willpotentially enable the utilisation of the understanding of epigenetic changes linkedto ELS for the development of much-needed novel therapeutic strategies and biomarkerdiscovery.

The development of the dopamine input to the medial prefrontal cortex occurs during adolescence and is a process that is vulnerable to disruption by stimulant drugs such as amphetamine. We have previously linked the amphetamine-induced disruption of dopamine connectivity and prefrontal cortex maturation during adolescence to the downregulation of the Netrin-1 receptor, DCC, in dopamine neurons. However, how DCC expression in dopamine neurons is itself regulated is completely unknown. MicroRNA (miRNA) regulation of mRNA translation and stability is a prominent mechanism linking environmental events to changes in protein expression. Here, using male mice, we show that miR-218 is expressed in dopamine neurons and is a repressor of DCC. Whereas Dcc mRNA levels increase from early adolescence to adulthood, miR-218 exhibits the exact opposite switch, most likely maintaining postnatal Dcc expression. This dynamic regulation appears to be selective to Dcc since the expression of Robo 1, the other guidance cue receptor target of miR-218, does not vary with age. Amphetamine in adolescence, but not in adulthood, increases miR-218 in the VTA and this event is required for drug-induced downregulation of Dcc mRNA and protein expression. This effect seems to be specific to Dcc because amphetamine does not alter Robo1. Furthermore, the upregulation of miR-218 by amphetamine requires dopamine D2 receptor activation. These findings identify miR-218 as regulator of DCC in the VTA both in normal development and after drug exposure in adolescence.

A Correction to this paper has been published: https://doi.org/10.1038/s41593-021-00848-y.