Bidirectional Relationship between Opioids and Disrupted Sleep: Putative Mechanisms.

Millions of Americans suffer from opiate use disorder, and over 100 die every day from opioid overdoses. Opioid use often progresses into a vicious cycle of abuse and withdrawal, resulting in very high rates of relapse. Although the physical and psychologic symptoms of opiate withdrawal are well-documented, sleep disturbances caused by chronic opioid exposure and withdrawal are less well-understood. These substances can significantly disrupt sleep acutely and in the long term. Yet poor sleep may influence opiate use, suggesting a bidirectional feed-forward interaction between poor sleep and opioid use. The neurobiology of how opioids affect sleep and how disrupted sleep affects opioid use is not well-understood. Here, we will summarize what is known about the effects of opioids on electroencephalographic sleep in humans and in animal models. We then discuss the neurobiology interface between reward-related brain regions that mediate arousal and wakefulness as well as the effect of opioids in sleep-related brain regions and neurotransmitter systems. Finally, we summarize what is known of the mechanisms underlying opioid exposure and sleep. A critical review of such studies, as well as recommendations of studies that evaluate the impact of manipulating sleep during withdrawal, will further our understanding of the cyclical feedback between sleep and opioid use. SIGNIFICANCE STATEMENT: We review recent studies on the mechanisms linking opioids and sleep. Opioids affect sleep, and sleep affects opioid use; however, the biology underlying this relationship is not understood. This review compiles recent studies in this area that fill this gap in knowledge.

Evidence from mouse and man for a role of neuregulin 3 in nicotine dependence.

Addiction to nicotine and the ability to quit smoking are influenced by genetic factors. We used functional genomic approaches (chromatin immunoprecipitation (ChIP) and whole-genome sequencing) to identify cAMP response element-binding protein (CREB) targets following chronic nicotine administration and withdrawal (WD) in rodents. We found that chronic nicotine and WD differentially modulate CREB binding to the gene for neuregulin 3 (NRG3). Quantitative analysis of saline, nicotine and nicotine WD in two biological replicates corroborate this finding, with NRG3 increases in both mRNA and protein following WD from chronic nicotine treatment. To translate these data for human relevance, single-nucleotide polymorphisms (SNPs) across NRG3 were examined for association with prospective smoking cessation among smokers of European ancestry treated with transdermal nicotine in two independent cohorts. Individual SNP and haplotype analysis support the association of NRG3 SNPs and smoking cessation success. NRG3 is a neural-enriched member of the epidermal growth factor family, and a specific ligand for the receptor tyrosine kinase ErbB4, which is also upregulated following nicotine treatment and WD. Mice with significantly reduced levels of NRG3 or pharmacological inhibition of ErbB4 show similar reductions in anxiety following nicotine WD compared with control animals, suggesting a role for NRG3 in nicotine dependence. Although the function of the SNP in NRG3 in humans is not known, these data suggest that Nrg3/ErbB4 signaling may be an important factor in nicotine dependence.

Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons.

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. CREB-mediated gene expression was both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 was activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. Overexpression of Bcl-2 reduced the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors.

Reduction of morphine abstinence in mice with a mutation in the gene encoding CREB.

Chronic morphine administration induces an up-regulation of several components of the cyclic adenosine 5'-monophosphate (cAMP) signal transduction cascade. The behavioral and biochemical consequences of opiate withdrawal were investigated in mice with a genetic disruption of the alpha and Delta isoforms of the cAMP-responsive element-binding protein (CREB). In CREBalphadelta mutant mice the main symptoms of morphine withdrawal were strongly attenuated. No change in opioid binding sites or in morphine-induced analgesia was observed in these mutant mice, and the increase of adenylyl cyclase activity and immediate early gene expression after morphine withdrawal was normal. Thus, CREB-dependent gene transcription is a factor in the onset of behavioral manifestations of opiate dependence.

Transgenerational inheritance of chronic adolescent stress: Effects of stress response and the amygdala transcriptome.

Adolescent stress can impact health and well-being not only during adulthood of the exposed individual but even in future generations. To investigate the molecular mechanisms underlying these long-term effects, we exposed adolescent males to stress and measured anxiety behaviors and gene expression in the amygdala-a critical region in the control of emotional states-in their progeny for two generations, offspring and grandoffspring. Male C57BL/6 mice underwent chronic unpredictable stress (CUS) for 2 weeks during adolescence and were used to produce two generations of offspring. Male and female offspring and grandoffspring were tested in behavioral assays to measure affective behavior and stress reactivity. Remarkably, transgenerational inheritance of paternal stress exposure produced a protective phenotype in the male, but not the female lineage. RNA-seq analysis of the amygdala from male offspring and grandoffspring identified differentially expressed genes (DEGs) in mice derived from fathers exposed to CUS. The DEGSs clustered into numerous pathways, and the "notch signaling" pathway was the most significantly altered in male grandoffspring. Therefore, we show that paternal stress exposure impacts future generations which manifest in behavioral changes and molecular adaptations.

Musashi, a neural RNA-binding protein required for Drosophila adult external sensory organ development.

A family of neural RNA-binding proteins has recently been described in both vertebrates and invertebrates. We have identified a new member of this family, the Drosophila musashi (msi) locus, which is required for development of adult external sensory organs (sensilla). In contrast with wild-type sensilla, which contain two outer support cells, the msi mutation typically results in the appearance of extra outer support cells. The msi putative RNA-binding protein is localized to the nucleus and appears to be expressed in all cells in each sensillum and predominantly in neurons during embryogenesis. We propose that the msi protein regulates sensillum development by controlling the expression of target genes at the posttranscriptional level.

The role of transcription factors cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER) in epileptogenesis.

Alterations in the brain that contribute to the development of epilepsy, also called epileptogenesis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER), in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein are increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing approximately threefold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy.

Spaced training induces normal long-term memory in CREB mutant mice.

BACKGROUND: The cAMP responsive element binding protein (CREB) is a transcription factor the activity of which is modulated by increases in the intracellular levels of cAMP and calcium. Results from studies with Aplysia, Drosophila and mice indicate that CREB-activated transcription is required for long-term memory. Furthermore, a recent study found that long-term memory for olfactory conditioning can be induced with a single trial in transgenic Drosophila expressing a CREB activator, whereas in normal flies, with presumably lower CREB-mediated transcription levels, conditioning requires multiple spaced trials. This suggests that CREB-mediated transcription is important in determining the type of training required for long-term memory of olfactory conditioning in Drosophila. Interestingly, studies with cultured Aplysia neurons indicated that removing a CREB repressor promoted the formation of long-term facilitation, a cellular model of non-associative memory. RESULTS: Here, we have confirmed that mice lacking the alpha and Delta CREB proteins (CREBalphaDelta-) have abnormal long-term, but not short-term, memory, as tested in an ethologically meaningful task. Importantly, additional spaced training can overcome the profound memory deficits of CREBalphaDelta- mutants. Increasing the intertrial interval from 1 to 60 minutes overcame the memory deficits of the CREBalphaDelta- mice in three distinct behavioral tasks: contextual fear conditioning, spatial learning and socially transmitted food preferences. CONCLUSIONS: Previous findings and results presented here demonstrate that CREB mutant mice have profound long-term memory deficits. Importantly, our findings indicate that manipulations of CREB function can affect the number of trials and the intertrial interval required for committing information to long-term memory. Remarkably, this effect of CREB function is not restricted to simple conditioning tasks, but also affects complex behaviours such as spatial memory and memory for socially transmitted food preferences.

Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse.

Addiction is a complex process that relies on the ability of an organism to integrate positive and negative properties of drugs of abuse. Therefore, studying the reinforcing as well as aversive components of drugs of abuse in a single model system will enable us to understand the role of final common mediators, such as cAMP response element-binding protein (CREB), in the addiction process. To this end, we analyzed mice with a mutation in the alpha and Delta isoforms of the CREB gene. Previously we have shown that CREB(alphaDelta) mutant mice in a mixed genetic background show attenuated signs of physical dependence, as measured by the classic signs of withdrawal. We have generated a uniform genetically stable F1 hybrid (129SvEv/C57BL/6) mouse line harboring the CREB mutation. We have found the functional activity of CREB in these F1 hybrid mice to be dramatically reduced compared with their wild-type littermates. These mice maintain a reduced withdrawal phenotype after chronic morphine. We are now poised to examine a number of complex behavioral phenotypes related to addiction in a well defined CREB-deficient mouse model. We demonstrate that the aversive properties of morphine are still present in CREB mutant mice despite a reduction of physical withdrawal. On the other hand, these mice do not respond to the reinforcing properties of morphine in a conditioned place preference paradigm. In contrast, CREB mutant mice demonstrate an enhanced response to the reinforcing properties of cocaine compared with their wild-type controls in both conditioned place preference and sensitization behaviors. These data may provide the first paradigm for differential vulnerability to various drugs of abuse.

Cyclic AMP response element-binding protein is required for normal maternal nurturing behavior.

Analysis of mice with targeted disruptions of fosB or the gene encoding dopamine beta-hydroxylase suggests that FosB and adrenergic signaling play critical roles in maternal nurturing behavior. The majority of neonates born to null females from either mutation fail to thrive, and virgin mutant females of both lines exhibit impaired pup retrieval. Considering whether FosB and adrenergic signaling might share a signaling pathway important for maternal behavior, we examined the role of a potential intermediary, cyclic AMP response element-binding protein (CREB). Here we report that approximately 40% of neonates (all heterozygous) born to mice lacking the major isoforms of CREB (Creb-alphaDelta-/-) died within several days of birth. In contrast, heterozygotes born to Creb-alphaDelta+/- females thrived. Cross-fostering demonstrated that neonates born to Creb-alphaDelta(-/dagger/-) females thrived when reared by wild-type females, and that Creb-alphaDelta-/- females were capable of rearing neonates whose maternal care was initiated by wild-type females. Further, virgin Creb-alphaDelta-/- females were deficient in pup retrieval despite exhibiting normal investigation of pups and of novel objects. No maternal behavior phenotype was present in mice with a null mutation of the cyclic AMP response element modulator (Crem) gene. Interestingly, the number of cells immunostaining for phospho-CREB (on Ser(133)) in the medial preoptic area of the hypothalamus, a key region for the expression of maternal behavior, increased nearly three-fold in wild-type mice following exposure to pups but not to novel objects. On the other hand, basal expression and induction of FosB in response to pup exposure appeared to be independent of CREB because levels were equivalent between wild-type and Creb-alphaDelta-/- females. These results implicate CREB in maternal nurturing behavior and suggest that CREB is not critical for expression or induction of FosB in adult virgin female mice.

Genetic analysis of drug addiction: the role of cAMP response element binding protein.

Many drugs of abuse, administered repeatedly over time, cause physical dependence which is expressed by a withdrawal syndrome when the drug is removed from the system. These processes can be thought of as adaptations of the neuronal system to an altered pharmacological state. The molecular mechanisms underlying these adaptations are still not known. A considerable amount of evidence is accumulating which implicates alterations in several components of the cAMP signal transduction cascade in these drug-induced processes. The transcription factor cAMP response element binding protein (CREB) in particular has been shown both in vitro and in vivo to be altered in response to several drugs of abuse, including opiates. This review discusses in detail this transcription factor and demonstrates its importance in the signal transduction cascades involving abused substances.

The mouse nac1 gene, encoding a cocaine-regulated Bric-a-brac Tramtrac Broad complex/Pox virus and Zinc finger protein, is regulated by AP1.

NAC1 cDNA was identified as a novel transcript induced in the nucleus accumbens from rats chronically treated with cocaine. NAC1 is a member of the Bric-a-brac Tramtrac Broad complex/Pox virus and Zinc finger family of transcription factors and has been shown by overexpression studies to prevent the development of behavioral sensitization resulting from repeated cocaine treatment. This paper reports the cloning and characterization of the corresponding gene. The mouse Nac1 gene consist of six exons, with exon 2 containing an alternative splice donor, providing a molecular explanation of the splice variants observed in mouse and rat. Transcripts of Nac1 were ubiquitously detected in different mouse tissues with prominent expression in the brain. The mouse Nac1 gene was localized to chromosome 8, suggesting a highly plausible candidate gene to explain differences in cocaine-induced behaviors between C57BL6/J and DBA/2J mice that had previously been mapped to the area. In addition, a functional AP1 binding site has been identified in an intron 1 enhancer of the Nac1 gene that plays an essential role in the activation of the gene in differentiation of neuroblastoma cells. Co-transfection with c-jun and c-fos expression plasmids, which encode the two subunits of AP1, activated the wild type Nac1 intron 1 enhancer two-fold over basal, nearly at the level of NAC1 enhancer activity seen in differentiated N2A cells. Mutation of the AP1 site completely abrogated all activation of the NAC1 enhancer in differentiated N2A cells. Activation of immediate early genes such as c-fos and c-jun following chronic drug treatments has been well characterized. The present data describe one potential regulatory cascade involving these transcription factors and activation of NAC1. Identification of drug induced alterations in gene expression is key to understanding the types of molecular adaptations underlying addiction.

Chronic reserpine administration selectively up-regulates beta 1- and alpha 1b-adrenergic receptors in rat brain: an autoradiographic study.

Rats were treated for 15 days with reserpine or vehicle. One day after the last treatment, animals were killed and frozen brain sections were prepared for in vitro autoradiography. Binding to beta-adrenergic receptors was measured with [125I]iodocyanopindolol, and binding selective for beta 1 and beta 2 subtypes was assessed by including non-radioactive drugs that selectively mask beta receptor subtypes. Total alpha 1-adrenergic receptor binding was measured with [3H]prazosin, while alpha 1a binding was measured with [3H]WB4101 (in the presence of unlabeled serotonin). Quantitative densitometric analysis revealed that chronic reserpine treatment caused an increase in beta binding throughout the brain, including the cortex, thalamus, amygdala, hippocampus, caudate-putamen and hypothalamus. This effect of reserpine was entirely confined to the beta 1 subtype in all regions examined. [3H]Prazosin binding (alpha 1a plus alpha 1b) was also increased after chronic reserpine in several regions of the cortex and thalamus, as well as the ventral hippocampus and caudal amygdala. No effect of chronic reserpine was seen on [3H]WB4101 binding, indicating that the effect of reserpine on alpha 1 receptors is limited to the alpha 1b subtype. The increase in alpha 1b binding after reserpine administration in rats was generally smaller and less widespread than that seen with beta 1 binding. Thus the effect of reserpine upon noradrenergic neurotransmission demonstrates a high degree of receptor specificity and regional selectivity.

Molecular genetic analysis of glucocorticoid signalling in development.

A null mutation of the glucocorticoid receptor was generated by homologous recombination. Mutant newborn mice showed impaired lung development, hypertrophy of the adrenal cortex and a strongly reduced size of the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT) was undetectable in the adrenals of the mutant mice. Serum levels of corticosterone were moderately and ACTH levels were strongly elevated in the mutants. A weaker but significant increase of corticosterone and ACTH was observed already in heterozygous animals. This points to a dysregulation of the HPA axis due to defective feedback regulation via the glucocorticoid receptor. Liver gluconeogenetic enzymes were reduced to a variable degree. Whereas survival of heterozygous mutants was not affected, most of the homozygous mutant mice died during the perinatal period.

Expression of the mouse glucocorticoid receptor and its role during development.

Genes encoding enzymes involved in gluconeogenesis are activated in liver shortly after birth by the synergistic effect of glucagon and glucocorticoids. This induction is achieved by the synergistic action of hormone responsive and liver-specific enhancer elements. In the case of glucocorticoids, this enhancer is composed of a glucocorticoid-response element (GRE) and a number of cell-specific hepatocyte nuclear factor 3 (HNF-3) binding sites. The GRE binds the ligand-activated glucocorticoid receptor (GR) which is ubiquitously expressed and the HNF-3 element binds a cell-specific protein factor. To further understand the role of cell-specific glucocorticoid signalling in the perinatal period and earlier during development we have studied the expression of the mouse GR gene. The gene has been cloned and fully characterized. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected back to day 9.5 of mouse development. The role of GR during mouse development has been further analysed by disruption of the GR gene in vivo by homologous recombination in mouse embryonic stem cells.

Effects of kainic acid induced seizures on immediate early gene expression in mice with a targeted mutation of the CREB gene.

The present study examined the response of immediate early genes following kainic acid induced seizures in mice lacking the alpha and delta isoforms of CREB. mRNA levels for c-fos, c-jun, and Krox-24 were measured following limbic seizure activity and were found to be induced in wild type as well as CREB mutant mice. This effect was also seen for these three mRNAs at the protein level as well as for FOS-B. Furthermore the time course of expression of FOS, JUN, KROX-24, and FOS-B proteins were essentially the same in CREB mutant mice as compared to wild-type controls. These data suggest that CREB alpha and delta are not required for the induction of immediate early genes following pharmacologically induced seizures.

Electroconvulsive shocks increase the concentration of neocortical and hippocampal neuropeptide Y (NPY)-like immunoreactivity in the rat.

Studies on humans and rats have suggested that neuropeptide Y (NPY) is involved in major depression and anxiety. Therefore, we conducted the present study in order to elucidate the effect of repeated (13 or 14 days) treatment of rats with electroconvulsive shocks (ECS) on the concentration of NPY-like immunoreactivity (-LI) in various brain regions, adrenals and plasma. In addition, the effect of ECS on 125I-NPY binding was studied in 3 brain regions. The effects of ECS were compared to effects of 3 control treatments: one group not being handled at all during the time period, one group handled like the ECS-group but not receiving shocks, and one group receiving shocks below the threshold for induction of convulsions. The latter group developed behavioral signs reminiscent of the inescapable shock-induced 'learned helplessness' syndrome (a proposed animal model of depression). We found that the concentration of NPY-LI in the frontal and parietal cortex and in the hippocampus were approximately doubled in the ECS-group as compared to the 3 control groups. No changes in NPY-LI were detected in the striatum, hypothalamus, pons, olfactory bulbs or cerebellum, nor in plasma or adrenals. In spite of the marked changes in NPY-LI concentration, the binding characteristics of 125I-NPY in the frontal and parietal cortex and in the hippocampus were similar in all 4 groups of rats. Finally, we confirmed the previous observation that ECS increase [3H]prazosin binding in cortex. In conclusion, ECS treatment increases neocortical and hippocampal NPY-LI concentrations, while leaving 125I-NPY binding unaffected. Subconvulsive shocks were without effect.

Electroconvulsive shock and reserpine increase alpha 1-adrenoceptor binding sites but not norepinephrine-stimulated phosphoinositide hydrolysis in rat brain.

Repeated treatment of rats with either electroconvulsive shock or reserpine increased the density of alpha 1-adrenoceptor binding sites labeled by [3H]prazosin in rat frontal cerebral cortex. In contrast, norepinephrine-stimulated phosphoinositide hydrolysis, which is mediated by alpha 1-adrenoceptors, was not significantly affected by either treatment. These data suggest that electroconvulsive shock and reserpine might increase only or predominantly a subtype of alpha 1-adrenoceptor that is not coupled to phosphoinositide hydrolysis.

Electroconvulsive shock but not antidepressant drugs increases alpha 1-adrenoceptor binding sites in rat brain.

Treatment of rats with electroconvulsive shock once daily for 10-12 days increased the density of alpha 1-adrenoceptors labeled by [3H]prazosin in homogenates of frontal cerebral cortex. A single treatment did not affect [3H]prazosin binding. Repeated treatment with electroconvulsive shock did not appear to affect alpha 1-adrenoceptor binding in the hippocampus or hypothalamus. Treatment of rats with reserpine also increased [3H]prazosin binding in the frontal cortex. In contrast to electroconvulsive shock, three weeks administration of tricyclic antidepressant drugs, monoamine oxidase inhibitors, or atypical antidepressant drugs did not significantly affect [3H]prazosin binding sites in the frontal cortex. The affinities of antidepressant drugs for alpha 1-adrenoceptors ranged from about 50 nM for tricyclic and atypical antidepressants to about 100 microM for monoamine oxidase inhibitors. The high affinities of the tricyclic and atypical antidepressant drugs for alpha 1-adrenoceptors could contribute to clinical differences between these classes of drugs and monoamine oxidase inhibitors. Furthermore, the electroconvulsive shock-induced increase in alpha 1-adrenoceptors could contribute to differences in clinical effects between this treatment and antidepressant drugs.

Analysis of the cAMP response on liver-specific gene expression in transgenic mice.

Expression of many genes is modulated by intracellular variations of cyclic AMP (cAMP) levels in response to different signals from the environment. This regulation is mediated via a cAMP-response element (CRE). This report addresses the role of cAMP in the physiological activation of a subset of liver-specific genes which are perinatally activated. The tyrosine aminotransferase (TAT) gene and other genes such as phosphoenolpyruvate carboxyquinase (PEPCK) and glucose-6-phosphatase, involved in gluconeogenesis, belong to this category. CRE elements derived from the rat TAT-3.6 kb enhancer have been positioned in chimeric constructs, such that the activity of the reporter gene LacZ is dependent on cAMP. The tissue-specificity of these constructs is guaranteed by the presence of the liver-specific enhancers of the alpha fetoprotein gene. These constructs have been tested in cells and transgenic mice demonstrating cAMP regulation, liver-specific expression and perinatal activation of the reporter gene. The CRE is recognized by a number of related proteins of which the cAMP-response element-binding factor (CREB) has been best studied. To assess the role of CREB in the in vivo transduction of cAMP signalling, mice deficient in CREB protein have been generated by homologous recombination in embryonic stem (ES) cells. Homozygous mutant mice, although recovering at a lower ratio than expected, do not display impairment of growth or development. The cAMP-dependent LacZ transgenic mice in a CREB mutant genetic background also show perinatal activation of the reporter gene.(ABSTRACT TRUNCATED AT 250 WORDS)