Mu-opioid receptor-expressing neurons in the paraventricular thalamus modulate chronic morphine-induced wake alterations.

Disrupted sleep is a symptom of many psychiatric disorders, including substance use disorders. Most drugs of abuse, including opioids, disrupt sleep. However, the extent and consequence of opioid-induced sleep disturbance, especially during chronic drug exposure, is understudied. We have previously shown that sleep disturbance alters voluntary morphine intake. Here, we examine the effects of acute and chronic morphine exposure on sleep. Using an oral self-administration paradigm, we show that morphine disrupts sleep, most significantly during the dark cycle in chronic morphine, with a concomitant sustained increase in neural activity in the Paraventricular Nucleus of the Thalamus (PVT). Morphine binds primarily to Mu Opioid Receptors (MORs), which are highly expressed in the PVT. Translating Ribosome Affinity Purification (TRAP)-Sequencing of PVT neurons that express MORs showed significant enrichment of the circadian entrainment pathway. To determine whether MOR + cells in the PVT mediate morphine-induced sleep/wake properties, we inhibited these neurons during the dark cycle while mice were self-administering morphine. This inhibition decreased morphine-induced wakefulness but not general wakefulness, indicating that MORs in the PVT contribute to opioid-specific wake alterations. Overall, our results suggest an important role for PVT neurons that express MORs in mediating morphine-induced sleep disturbance.

A novel Oprm1-Cre mouse maintains endogenous expression, function and enables detailed molecular characterization of µ-opioid receptor cells.

Key targets of both the therapeutic and abused properties of opioids are µ-opioid receptors (MORs). Despite years of research investigating the biochemistry and signal transduction pathways associated with MOR activation, we do not fully understand the cellular mechanisms underlying opioid addiction. Given that addictive opioids such as morphine, oxycodone, heroin, and fentanyl all activate MORs, and current therapies such as naloxone and buprenorphine block this activation, the availability of tools to mechanistically investigate opioid-mediated cellular and behavioral phenotypes are necessary. Therefore, we derived, validated, and applied a novel MOR-specific Cre mouse line, inserting a T2A cleavable peptide sequence and the Cre coding sequence into the MOR 3'UTR. Importantly, this line shows specificity and fidelity of MOR expression throughout the brain and with respect to function, there were no differences in behavioral responses to morphine when compared to wild type mice, nor are there any alterations in Oprm1 gene expression or receptor density. To assess Cre recombinase activity, MOR-Cre mice were crossed with the floxed GFP-reporters, RosaLSLSun1-sfGFP or RosaLSL-GFP-L10a. The latter allowed for cell type specific RNA sequencing via TRAP (Translating Ribosome Affinity Purification) of striatal MOR+ neurons following opioid withdrawal. The breadth of utility of this new tool will greatly facilitate the study of opioid biology under varying conditions.

Chronic Sleep Deprivation Blocks Voluntary Morphine Consumption but Not Conditioned Place Preference in Mice.

The opioid epidemic remains a significant healthcare problem and is attributable to over 100,000 deaths per year. Poor sleep increases sensitivity to pain, impulsivity, inattention, and negative affect, all of which might perpetuate drug use. Opioid users have disrupted sleep during drug use and withdrawal and report poor sleep as a reason for relapse. However, preclinical studies investigating the relationship between sleep loss and substance use and the associated underlying neurobiological mechanisms of potential interactions are lacking. One of the most common forms of sleep loss in modern society is chronic short sleep (CSS) (<7 h/nightly for adults). Here, we used an established model of CSS to investigate the influence of disrupted sleep on opioid reward in male mice. The CSS paradigm did not increase corticosterone levels or depressive-like behavior after a single sleep deprivation session but did increase expression of Iba1, which typically reflects microglial activation, in the hypothalamus after 4 weeks of CSS. Rested control mice developed a morphine preference in a 2-bottle choice test, while mice exposed to CSS did not develop a morphine preference. Both groups demonstrated morphine conditioned place preference (mCPP), but there were no differences in conditioned preference between rested and CSS mice. Taken together, our results show that recovery sleep after chronic sleep disruption lessens voluntary opioid intake, without impacting conditioned reward associated with morphine.

Adenosine Monophosphate-activated Protein Kinase (AMPK) in serotonin neurons mediates select behaviors during protracted withdrawal from morphine in mice.

Serotonin neurotransmission has been implicated in behavior deficits that occur during protracted withdrawal from opioids. In addition, studies have highlighted multiple pathways whereby serotonin (5-HT) modulates energy homeostasis, however the underlying metabolic effects of opioid withdrawal have not been investigated. A key metabolic regulator that senses the energy status of the cell and regulates fuel availability is Adenosine Monophosphate-activated Protein Kinase (AMPK). To investigate the interaction between cellular metabolism and serotonin in modulating protracted abstinence from morphine, we depleted AMPK in serotonin neurons. Morphine exposure via drinking water generates dependence in these mice, and both wildtype and serotonergic AMPK knockout mice consume similar amounts of morphine with no changes in body weight. Serotonergic AMPK contributes to baseline differences in open field and social interaction behaviors and blocks abstinence induced reductions in immobility following morphine withdrawal in the tail suspension test. Lastly, morphine locomotor sensitization is blunted in mice lacking AMPK in serotonin neurons. Taken together, our results suggest serotonergic AMPK mediates both baseline and protracted morphine withdrawal-induced behaviors.

Interleukin-1α in the ventral hippocampus increases stress vulnerability and inflammation-related processes.

Mechanisms of stress vulnerability remain elusive. Previous research demonstrated that inflammation-related processes in the brain play a role in stress vulnerability. Our previous research showed that inflammatory processes in the ventral hippocampus (vHPC) induced a stress vulnerable phenotype. To further understand neuroinflammatory processes in the vHPC in stressed rats, we determined that protein levels of the pro-inflammatory cytokine interleukin-1-α (IL-1α), but not interleukin-1ß (IL-1ß), were increased in the vHPC of rats vulnerable to the effects of repeated social defeat compared to rats resilient to its effects. Injections of IL-1α into the vHPC increased stress vulnerability as characterized by increases in passive coping during defeat and subsequent decreased social interactions. Conversely, injections of recombinant IL-1 receptor antagonist (IL1-RA) increased latencies to social defeat and decreased anxiety-like behaviors during social interaction, suggesting an reduction in stress vulnerability. Protein analyses revealed increased FosB expression in the vHPC of IL-1α-injected rats, and increased HPA activation following a social encounter. Further analysis of vHPC of IL1-α-injected rats showed increased density of microglia, increased expression of the pro-inflammatory cytokine HMGB1, and increases in a marker for vascular remodeling. Taken together, these data show increasing IL-1α during stress exposure is sufficient to produce a stress vulnerable phenotype potentially by increasing inflammation-related processes in the vHPC.LAY SUMMARYOur previous research demonstrated that inflammation-related processes in the brain play a role in inducing vulnerability to the effects of repeated social stress in rats. Here we demonstrate that a pro-inflammatory cytokine interleukin-1-α (IL-1α) induces inflammatory processes in the vHPC and behavioral vulnerability in stressed rats, whereas blocking IL receptors produces the opposite effects on behavioral vulnerability. Together, these results identify a substrate in the vHPC that produces vulnerability to stress by increasing inflammation-related processes in the vHPC.

The gut microbiome regulates the increases in depressive-type behaviors and in inflammatory processes in the ventral hippocampus of stress vulnerable rats.

Chronic exposure to stress is associated with increased incidence of depression, generalized anxiety, and PTSD. However, stress induces vulnerability to such disorders only in a sub-population of individuals, as others remain resilient. Inflammation has emerged as a putative mechanism for promoting stress vulnerability. Using a rodent model of social defeat, we have previously shown that rats with short-defeat latencies (SL/vulnerable rats) show increased anxiety- and depression-like behaviors, and these behaviors are mediated by inflammation in the ventral hippocampus. The other half of socially defeated rats show long-latencies to defeat (LL/resilient) and are similar to controls. Because gut microbiota are important activators of inflammatory substances, we assessed the role of the gut microbiome in mediating vulnerability to repeated social defeat stress. We analyzed the fecal microbiome of control, SL/vulnerable, and LL/resilient rats using shotgun metagenome sequencing and observed increased expression of immune-modulating microbiota, such as Clostridia, in SL/vulnerable rats. We then tested the importance of gut microbiota to the SL/vulnerable phenotype. In otherwise naive rats treated with microbiota from SL/vulnerable rats, there was higher microglial density and IL-1ß expression in the vHPC, and higher depression-like behaviors relative to rats that received microbiota from LL/resilient rats, non-stressed control rats, or vehicle-treated rats. However, anxiety-like behavior during social interaction was not altered by transplant of the microbiome of SL/vulnerable rats into non-stressed rats. Taken together, the results suggest the gut microbiome contributes to the depression-like behavior and inflammatory processes in the vHPC of stress vulnerable individuals.

CREB deletion increases resilience to stress and downregulates inflammatory gene expression in the hippocampus.

The transcription factor CREB (cyclic AMP response element (CRE)-binding protein) is implicated in the pathophysiology and treatment of depression. Structural and functional studies in both animals and humans suggest that abnormalities of the hippocampus may play a role in depression. CREB regulates thousands of genes, yet to date, only a handful that mediate depression or antidepressant response have been identified as relevant CREB targets. In order to comprehensively identify genes regulated by CREB in the hippocampus, we employed translating ribosome affinity purification (TRAP) to detect actively translating mRNAs in wild type and CREB-deficient mice. Using CrebloxP/loxP; RosaLSL-GFP-L10a mice, we conducted whole genome sequencing to identify transcripts only in cells that lack CREB, as introduction of Cre-recombinase simultaneously deleted CREB and expressed GFP-tagged L10a ribosomes that enabled TRAP. We identified over 200 downregulated genes predominantly associated with inflammation and the immune system, including toll-like receptor 1 (TLR1). To determine if baseline disruption in gene expression in the hippocampus of CREB-deficient mice can modulate behavior, we used unpredictable chronic mild stress (UCMS) to produce a set of behavioral alterations with strong validity for depression. We found that CREB-deficient mice demonstrated resilience to the physiological effects of UCMS and also showed changes in affective behaviors specifically in the presence of stress. TLR1 expression was increased following UCMS in control but not in CREB-deficient mice. The results suggest that CREB-mediated regulation of immune system and inflammatory factors may provide additional targets for the treatment of depression.

Orexin signaling during social defeat stress influences subsequent social interaction behaviour and recognition memory.

Orexins are neuropeptides synthesized in the lateral hypothalamus that influence arousal, feeding, reward pathways, and the response to stress. However, the role of orexins in repeated stress is not fully characterized. Here, we examined how orexins and their receptors contribute to the coping response during repeated social defeat and subsequent anxiety-like and memory-related behaviors. Specifically, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to stimulate orexins prior to each of five consecutive days of social defeat stress in adult male rats. Additionally, we determined the role of the orexin 2 receptor in these behaviors by using a selective orexin 2 receptor antagonist (MK-1064) administered prior to each social defeat. Following the 5 day social defeat conditioning period, rats were evaluated in social interaction and novel object recognition paradigms to assess anxiety-like behavior and recognition memory, respectively. Activation of orexin neurons by DREADDs prior to each social defeat decreased the average latency to become defeated across 5 days, indicative of a passive coping strategy that we have previously linked to a stress vulnerable phenotype. Moreover, stimulation of orexin signaling during defeat conditioning decreased subsequent social interaction and performance in the novel object recognition test indicating increased subsequent anxiety-like behavior and reduced recognition memory. Blocking the orexin 2 receptor during repeated defeat did not alter these effects. Together, our results suggest that orexin neuron activation produces a passive coping phenotype during social defeat leading to subsequent anxiety-like behaviors and memory deficits.

Reduced Orexin System Function Contributes to Resilience to Repeated Social Stress.

Exposure to stress increases the risk of developing affective disorders such as depression and post-traumatic stress disorder (PTSD). However, these disorders occur in only a subset of individuals, those that are more vulnerable to the effects of stress, whereas others remain resilient. The coping style adopted to deal with the stressor, either passive or active coping, is related to vulnerability or resilience, respectively. Important neural substrates that mediate responses to a stressor are the orexins. These neuropeptides are altered in the cerebrospinal fluid of patients with stress-related illnesses such as depression and PTSD. The present experiments used a rodent social defeat model that generates actively coping rats and passively coping rats, which we have previously shown exhibit resilient and vulnerable profiles, respectively, to examine if orexins play a role in these stress-induced phenotypes. In situ radiolabeling and qPCR revealed that actively coping rats expressed significantly lower prepro-orexin mRNA compared with passively coping rats. This led to the hypothesis that lower levels of orexins contribute to resilience to repeated social stress. To test this hypothesis, rats first underwent 5 d of social defeat to establish active and passive coping phenotypes. Then, orexin neurons were inhibited before each social defeat for three additional days using designer receptors exclusively activated by designer drugs (DREADDs). Inhibition of orexins increased social interaction behavior and decreased depressive-like behavior in the vulnerable population of rats. Indeed, these data suggest that lowering orexins promoted resilience to social defeat and may be an important target for treatment of stress-related disorders.

Orexin 2 receptor regulation of the hypothalamic-pituitary-adrenal (HPA) response to acute and repeated stress.

Orexins are hypothalamic neuropeptides that have a documented role in mediating the acute stress response. However, their role in habituation to repeated stress, and the role of orexin receptors (OX1R and OX2R) in the stress response, has yet to be defined. Orexin neuronal activation and levels in the cerebrospinal fluid (CSF) were found to be stimulated with acute restraint, but were significantly reduced by day five of repeated restraint. As certain disease states such as panic disorder are associated with increased central orexin levels and failure to habituate to repeated stress, the effect of activating orexin signaling via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) on the hypothalamic-pituitary-adrenal (HPA) response was evaluated after repeated restraint. While vehicle-treated rats displayed habituation of Adrenocorticotropic Hormone (ACTH) from day 1 to day 5 of restraint, stimulating orexins did not further increase ACTH beyond vehicle levels for either acute or repeated restraint. We delineated the roles of orexin receptors in acute and repeated stress using a selective OX2R antagonist (MK-1064). Pretreatment with MK-1064 reduced day 1 ACTH levels, but did not allow further habituation on day 5 compared with vehicle-treated rats, indicating that endogenous OX2R activity plays a role in acute stress, but not in habituation to repeated stress. However, in restrained rats with further stimulated orexins by DREADDs, MK-1064 decreased ACTH levels on day 5. Collectively, these results indicate that the OX2R plays a role in acute stress, and can prevent habituation to repeated stress under conditions of high orexin release.

Two weeks of predatory stress induces anxiety-like behavior with co-morbid depressive-like behavior in adult male mice.

Psychological stress can have devastating and lasting effects on a variety of behaviors, especially those associated with mental illnesses such as anxiety and depression. Animal models of chronic stress are frequently used to elucidate the mechanisms underlying the relationship between stress and mental health disorders and to develop improved treatment options. The current study expands upon a novel chronic stress paradigm for mice: predatory stress. The predatory stress model incorporates the natural predator-prey relationship that exists among rats and mice and allows for greater interaction between the animals, in turn increasing the extent of the stressful experience. In this study, we evaluated the behavioral effects of exposure to 15 days of predatory stress on an array of behavioral indices. Up to 2 weeks after the end of stress, adult male mice showed an increase of anxiety-like behaviors as measured by the open field and social interaction tests. Animals also expressed an increase in depressive-like behavior in the sucrose preference test. Notably, performance on the novel object recognition task, a memory test, improved after predatory stress. Taken as a whole, our results indicate that 15 exposures to this innovative predatory stress paradigm are sufficient to elicit robust anxiety-like behaviors with evidence of co-morbid depressive-like behavior, as well as changes in cognitive behavior in male mice.