Early resource scarcity causes cortical astrocyte enlargement and sex-specific changes in the orbitofrontal cortex transcriptome in adult rats.

Astrocyte morphology affects function, including the regulation of glutamatergic signaling. This morphology changes dynamically in response to the environment. However, how early life manipulations alter adult cortical astrocyte morphology is underexplored. Our lab uses brief postnatal resource scarcity, the limited bedding and nesting (LBN) manipulation, in rats. We previously found that LBN augments maternal behaviors and promotes later resilience to adult addiction-related behaviors, reducing impulsivity, risky decision-making, and morphine self-administration. These behaviors rely on glutamatergic transmission in the medial orbitofrontal (mOFC) and medial prefrontal (mPFC) cortex. Here we tested whether LBN changed astrocyte morphology in the mOFC and mPFC of adult rats using a novel viral approach that, unlike traditional markers, fully labels astrocytes. Prior exposure to LBN causes an increase in the surface area and volume of astrocytes in the mOFC and mPFC of adult males and females relative to control-raised rats. We next used bulk RNA sequencing of OFC tissue to assess transcriptional changes that could increase astrocyte size in LBN rats. LBN caused mainly sex-specific changes in differentially expressed genes. Pathway analysis revealed that OFC glutamatergic signaling is altered by LBN in males and females, but the gene changes in that pathway differed across sex. This may represent a convergent sex difference where glutamatergic signaling, which affects astrocyte morphology, is altered by LBN via sex-specific mechanisms. Collectively, these studies highlight that astrocytes may be an important cell type that mediates the effect of early resource scarcity on adult brain function.

Sex differences in pre- and post-synaptic glutamate signaling in the nucleus accumbens core.

BACKGROUND: Glutamate signaling within the nucleus accumbens underlies motivated behavior and is involved in psychiatric disease. Although behavioral sex differences in these processes are well-established, the neural mechanisms driving these differences are largely unexplored. In these studies, we examine potential sex differences in synaptic plasticity and excitatory transmission within the nucleus accumbens core. Further understanding of baseline sex differences in reward circuitry will shed light on potential mechanisms driving behavioral differences in motivated behavior and psychiatric disease. METHODS: Behaviorally naïve adult male and female Long-Evans rats, C57Bl/6J mice, and constitutive PKMζ knockout mice were killed and tissue containing the nucleus accumbens core was collected for ex vivo slice electrophysiology experiments. Electrophysiology recordings examined baseline sex differences in synaptic plasticity and transmission within this region and the potential role of PKMζ in long-term depression. RESULTS: Within the nucleus accumbens core, both female mice and rats exhibit higher AMPA/NMDA ratios compared to male animals. Further, female mice have a larger readily releasable pool of glutamate and lower release probability compared to male mice. No significant sex differences were detected in spontaneous excitatory postsynaptic current amplitude or frequency. Finally, the threshold for induction of long-term depression was lower for male animals than females, an effect that appears to be mediated, in part, by PKMζ. CONCLUSIONS: We conclude that there are baseline sex differences in synaptic plasticity and excitatory transmission in the nucleus accumbens core. Our data suggest there are sex differences at multiple levels in this region that should be considered in the development of pharmacotherapies to treat psychiatric illnesses such as depression and substance use disorder.

Understanding normal neural signaling within the nucleus accumbens, a key brain region involved in psychiatric disease including substance use disorder and depression, could provide insight into treatment options for these disorders. Although we know the behaviors regulated by the nucleus accumbens can differ between males and females, we do not understand the underlying differences in brain processing that could contribute to these behavioral differences. Further, even in cases when these behaviors are not different, the underlying brain signaling may exhibit sex-specific mechanisms. The current studies examined excitatory signaling with the nucleus accumbens in both rats and mice at the level of both individual cells and circuits. We found that female rodents (rats and mice) exhibit higher levels of excitatory signaling within the nucleus accumbens than male rodents. Further, procedures that can dampen neural transmission in males are not sufficient to do so in females, suggesting that excitatory signaling in the nucleus accumbens of females is less plastic. Finally, our last set of studies utilized mice missing the protein, PKMζ, and demonstrated that this reversed some of the sex differences seen in normal mice, pointing to a critical role for this protein in maintaining these differences. Our data suggest there are sex differences at multiple levels in this region that should be considered in the development of pharmacotherapies to treat psychiatric illnesses such as depression and substance use disorder.

Early resource scarcity causes cortical astrocyte enlargement and sex-specific changes in the orbitofrontal cortex transcriptome in adult rats.

Astrocyte morphology affects function, including the regulation of glutamatergic signaling. This morphology changes dynamically in response to the environment. However, how early life manipulations alter adult cortical astrocyte morphology is underexplored. Our lab uses brief postnatal resource scarcity, the limited bedding and nesting (LBN) manipulation, in rats. We previously found that LBN promotes later resilience to adult addiction-related behaviors, reducing impulsivity, risky decision-making, and morphine self-administration. These behaviors rely on glutamatergic transmission in the medial orbitofrontal (mOFC) and medial prefrontal (mPFC) cortex. Here we tested whether LBN changed astrocyte morphology in the mOFC and mPFC of adult rats using a novel viral approach that, unlike traditional markers, fully labels astrocytes. Prior exposure to LBN causes an increase in the surface area and volume of astrocytes in the mOFC and mPFC of adult males and females relative to control-raised rats. We next used bulk RNA sequencing of OFC tissue to assess transcriptional changes that could increase astrocyte size in LBN rats. LBN caused mainly sex-specific changes in differentially expressed genes. However, Park7, which encodes for the protein DJ-1 that alters astrocyte morphology, was increased by LBN across sex. Pathway analysis revealed that OFC glutamatergic signaling is altered by LBN in males and females, but the gene changes in that pathway differed across sex. This may represent a convergent sex difference where glutamatergic signaling, which affects astrocyte morphology, is altered by LBN via sex-specific mechanisms. Collectively, these studies highlight that astrocytes may be an important cell type that mediates the effect of early resource scarcity on adult brain function.

Paternal morphine exposure in rats reduces social play in adolescent male progeny without affecting drug-taking behavior in juvenile males or female offspring.

The ongoing opioid addiction crisis necessitates the identification of novel risk factors to improve prevention and treatment of opioid use disorder. Parental opioid exposure has recently emerged as a potential regulator of offspring vulnerability to opioid misuse, in addition to heritable genetic liability. An understudied aspect of this "missing heritability" is the developmental presentation of these cross-generational phenotypes. This is an especially relevant question in the context of inherited addiction-related phenotypes, given the prominent role of developmental processes in the etiology of psychiatric disorders. Paternal morphine self-administration was previously shown to alter the sensitivity to the reinforcing and antinociceptive properties of opioids in the next generation. Here, phenotyping was expanded to include the adolescent period, with a focus on endophenotypes related to opioid use disorders and pain. Paternal morphine exposure did not alter heroin or cocaine self-administration in male and female juvenile progeny. Further, baseline sensory reflexes related to pain were unaltered in morphine-sired adolescent rats of either sex. However, morphine-sired adolescent males exhibited a reduction in social play behavior. Our findings suggest that, in morphine-sired male offspring, paternal opioid exposure does not affect opioid intake during adolescence, suggesting that this phenotype does not emerge until later in life. Altered social behaviors in male morphine-sired adolescents indicate that the changes in drug-taking behavior in adults sired by morphine-exposed sires may be due to more complex factors not yet fully assessed.

Paternal morphine exposure enhances morphine self-administration and induces region-specific neural adaptations in reward-related brain regions of male offspring.

Background: A growing body of preclinical studies report that preconceptional experiences can have a profound and long-lasting impact on adult offspring behavior and physiology. However, less is known about paternal drug exposure and its effects on reward sensitivity in the next generation. Methods: Adult male rats self-administered morphine for 65 days; controls received saline. Sires were bred to drug-naïve dams to produce first-generation (F1) offspring. Morphine, cocaine, and nicotine self-administration were measured in adult F1 progeny. Molecular correlates of addiction-like behaviors were measured in reward-related brain regions of drug naïve F1 offspring. Results: Male, but not female offspring produced by morphine-exposed sires exhibited dose-dependent increased morphine self-administration and increased motivation to earn morphine infusions under a progressive ratio schedule of reinforcement. This phenotype was drug-specific as self-administration of cocaine, nicotine, and sucrose were not altered by paternal morphine history. The male offspring of morphine-exposed sires also had increased expression of mu-opioid receptors in the ventral tegmental area but not in the nucleus accumbens. Conclusions: Paternal morphine exposure increased morphine addiction-like behavioral vulnerability in male but not female progeny. This phenotype is likely driven by long-lasting neural adaptations within the reward neural brain pathways.

Early resource scarcity alters motivation for natural rewards in a sex- and reinforcer-dependent manner.

RATIONALE: Early life adversity impacts reward-related behaviors, including reward seeking for drugs of abuse. However, the effects of early stress on natural rewards, such as food and social rewards, which have strong implications for symptoms of psychiatric conditions such as major depressive disorder (MDD), are understudied. To fill this gap, we used the limited bedding and nesting (LBN) procedure to assess the impact of early resource scarcity on motivational drive for both food and social rewards in rats. METHODS: Male and female Long Evans rats were reared in either an LBN environment, with limited nesting materials and no enrichment, from their postnatal day 2-9 or control environment with ample nesting materials and enrichment. As adults, they were tested for reward-seeking behavior on progressive ratio operant tasks: food reward (sucrose) or social reward (access to a same-sex/age conspecific). RESULTS: We observed sex differences in the impact of LBN on motivation for natural rewards. In males, LBN increased motivation for both a sucrose and social reward. In females, LBN reduced motivation for sucrose but had no effect on social reward. CONCLUSIONS: These results suggest that the effects of LBN on motivation for natural rewards are both sex- and reinforcer-dependent, with males and females showing differential motivation for food and social rewards following early scarcity. Our previous data revealed an LBN-driven reduction in motivation for morphine in males and no effect in females, highlighting the reinforcer-dependent impact of early resource scarcity on motivated behavior more widely.

Multi-chemokine receptor antagonist RAP-103 inhibits opioid-derived respiratory depression, reduces opioid reinforcement and physical dependence, and normalizes opioid-induced dysregulation of mesolimbic chemokine receptors in rats.

Chemokine-opioid crosstalk is a physiological crossroads for influencing therapeutic and adverse effects of opioids. Activation of chemokine receptors, especially CCR2, CCR5 and CXCR4, reduces opioid-induced analgesia by desensitizing OPRM1 receptors. Chemokine receptor antagonists (CRAs) enhance opioid analgesia, but knowledge about how CRAs impact adverse opioid effects remains limited. We examined effects of RAP-103, a multi-CRA orally active peptide analog of "DAPTA", on opioid-derived dependence, reinforcement, and respiratory depression in male rats and on changes in chemokine and OPRM1 (µ opioid) receptor levels in mesolimbic substrates during opioid abstinence. In rats exposed to chronic morphine (75 mg pellet x 7 d), daily RAP-103 (1 mg/kg, IP) treatment reduced the severity of naloxone-precipitated withdrawal responses. For self-administration (SA) studies, RAP-103 (1 mg/kg, IP) reduced heroin acquisition (0.1 mg/kg/inf) and reinforcing efficacy (assessed by motivation on a progressive-ratio reinforcement schedule) but did not impact sucrose intake. RAP-103 (1-3 mg/kg, IP) also normalized the deficits in oxygen saturation and enhancement of respiratory rate caused by morphine (5 mg/kg, SC) exposure. Abstinence from chronic morphine elicited brain-region specific changes in chemokine receptor protein levels. CCR2 and CXCR4 were increased in the ventral tegmental area (VTA), whereas CCR2 and CCR5 were reduced in the nucleus accumbens (NAC). Effects of RAP-103 (1 mg/kg, IP) were focused in the NAC, where it normalized morphine-induced deficits in CCR2 and CCR5. These results identify CRAs as potential biphasic function opioid signaling modulators to enhance opioid analgesia and inhibit opioid-derived dependence and respiratory depression.

Paternal nicotine taking elicits heritable sex-specific phenotypes that are mediated by hippocampal Satb2.

Nicotine intake, whether through tobacco smoking or e-cigarettes, remains a global health concern. An emerging preclinical literature indicates that parental nicotine exposure produces behavioral, physiological, and molecular changes in subsequent generations. However, the heritable effects of voluntary parental nicotine taking are unknown. Here, we show increased acquisition of nicotine taking in male and female offspring of sires that self-administered nicotine. In contrast, self-administration of sucrose and cocaine were unaltered in male and female offspring suggesting that the intergenerational effects of paternal nicotine taking may be reinforcer specific. Further characterization revealed memory deficits and increased anxiety-like behaviors in drug-naive male, but not female, offspring of nicotine-experienced sires. Using an unbiased, genome-wide approach, we discovered that these phenotypes were associated with decreased expression of Satb2, a transcription factor known to play important roles in synaptic plasticity and memory formation, in the hippocampus of nicotine-sired male offspring. This effect was sex-specific as no changes in Satb2 expression were found in nicotine-sired female offspring. Finally, increasing Satb2 levels in the hippocampus prevented the escalation of nicotine intake and rescued the memory deficits associated with paternal nicotine taking in male offspring. Collectively, these findings indicate that paternal nicotine taking produces heritable sex-specific molecular changes that promote addiction-like phenotypes and memory impairments in male offspring.

Opioid and Sucrose Craving Are Accompanied by Unique Behavioral and Affective Profiles after Extended Abstinence in Male and Female Rats.

Incubation of craving refers to the intensification of drug-seeking behavior in response to reward-paired cues over the course of abstinence. In rodents, craving and drug-seeking behaviors have been measured by an increase in lever pressing in the absence of reinforcer availability in response to cue presentations. However, craving in rodents is difficult to define and little is known about the behavioral signatures that accompany increased drug-seeking behavior measured by lever pressing. The affective components of relapse are also important, but understudied in rodents. Hormonal fluctuations influence craving for psychostimulants, but little is known about the impact of the estrous cycle on opioid-seeking behavior. This study sought to delineate the behavioral and affective signatures associated with craving, and to examine the influence of the female estrous cycle on craving. Male and female rats underwent 10 d of intravenous opioid self-administration. Separate cohorts of control rats self-administered oral sucrose, a natural nondrug reward. Cue-induced seeking tests were conducted after 1 or 30d of forced abstinence. These sessions were recorded and scored for overall locomotion, instances of sniffing, grooming, or hyperactivity. Ultrasonic vocalizations (USVs) were also recorded to determine affective profiles that accompany opioid seeking. Although active lever presses and overall locomotion increased unanimously over extended abstinence from heroin and sucrose, a sex- and reinforcer-specific behavioral and affective signature of craving emerged. Furthermore, although the female estrous cycle did not affect taking or seeking, it appears to influence more granular behaviors.

Cocaine-Induced Changes in Sperm Cdkn1a Methylation Are Associated with Cocaine Resistance in Male Offspring.

Paternal environmental perturbations can influence the physiology and behavior of offspring. For example, our previous work showed reduced cocaine reinforcement in male, but not female, progeny of rat sires that self-administered cocaine. The information transfer from sire to progeny may occur through epigenetic marks in sperm, encompassing alterations in small noncoding RNAs, including microRNAs (miRNAs) and/or DNA methylation. Here, no reliable changes in miRNAs in the sperm of cocaine- relative to saline-experienced sires were identified. In contrast, 272 differentially methylated regions were observed in sperm between these groups. Two hypomethylated promoter regions in the sperm of cocaine-experienced rats were upstream of cyclin-dependent kinase inhibitor 1a (Cdkn1a). Cdkn1a mRNA also was selectively increased in the NAc of cocaine-sired male (but not female) offspring. Cocaine self-administration also enhanced Cdkn1a expression in the accumbens of cocaine-sired rats. These results suggest that changes in Cdkn1a may play a role in the reduced cocaine reinforcing efficacy observed in cocaine-sired male rats. Introducing a 90 d delay between sire self-administration and breeding reversed both cocaine resistance and the increase in accumbens Cdkn1a mRNA in male offspring, indicating that cocaine-induced epigenetic modifications are eliminated with sperm turnover. Collectively, our results indicate that cocaine self-administration produces hypomethylation of Cdkn1a in sperm and a selective increase in the expression of this gene in the NAc of male offspring, which is associated with blunted cocaine reinforcement.SIGNIFICANCE STATEMENT The relatively new field of transgenerational epigenetics explores the effects of environmental perturbations on offspring behavior and physiology. Our prior work in rats indicated that male, but not female, progeny of sires that self-administered cocaine displayed reduced cocaine reinforcement. The information transfer from sire to progeny may occur through heritable epigenetic marks in sperm, including DNA methylation. The present findings revealed two hypomethylated promoter regions upstream of the Cdkn1a gene in sire sperm. Remarkably, Cdkn1a expression was selectively decreased in offspring NAc, a brain region that regulates cocaine reinforcement.

microRNA expression levels in the nucleus accumbens correlate with morphine-taking but not morphine-seeking behaviour in male rats.

A powerful motivation to seek opioids remains after drug cessation and intensifies during extended periods of abstinence. Unfortunately, biomarkers associated with continued drug seeking have not been described. Moreover, previous studies have focused on the effects of early abstinence with little exploration into the long-term drug-induced mechanisms that occur after extended abstinence. Here we demonstrated that 30 days (D) of forced abstinence results in a time-dependent increase in morphine seeking in a rat model of morphine self-administration (SA). We measured expression of known drug-responsive microRNAs (miRNAs) in the nucleus accumbens, an area critical for reward-related plasticity, during early or late abstinence in animals that underwent either a cue-induced relapse test or no relapse test. miRNAs are small noncoding RNAs that represent suitable biomarker candidates due to their long-lasting nature. mir-32-5p levels during early abstinence negatively correlated with active lever pressing in both cue-exposed and cue-naïve animals. mir-1298-5p positively correlated with drug SA history after a relapse test during late abstinence. When animals underwent acute abstinence with no relapse test, mir-1298-5p correlated with drug infusions and active lever pressing during SA. In late abstinence with no relapse test, mir-137-3p negatively correlated with drug infusions. Regulation of mir-32-5p target genes and significant correlation of target gene mRNA with mir-32-5p was observed after abstinence. These results indicate that lasting regulation of miRNA expression is associated with drug intake following morphine SA. In addition, we conclude that the miRNA profile undergoes regulation from early to late abstinence and miRNA expression may indicate past drug history.

Effects of early life adversity on male reproductive behavior and the medial preoptic area transcriptome.

Early life adversity can alter reproductive development in humans, changing the timing of pubertal onset and sexual activity. One common form of early adversity is limited access to resources. This adversity can be modeled in rats using the limited bedding/nesting model (LBN), in which dams and pups are placed in a low resource environment from pups' postnatal days 2-9. Our laboratory previously found that adult male rats raised in LBN conditions have elevated levels of plasma estradiol compared to control males. In females, LBN had no effect on plasma hormone levels, pubertal timing, or estrous cycle duration. Estradiol mediates male reproductive behaviors. Thus, here we compared reproductive behaviors in adult males exposed to LBN vs. control housing. LBN males acquired the suite of reproductive behaviors (mounts, intromissions, and ejaculations) more quickly than their control counterparts over 3 weeks of testing. However, there was no effect of LBN in males on puberty onset or masculinization of certain brain regions, suggesting LBN effects on estradiol and reproductive behaviors manifest after puberty. In male and female rats, we next used RNA sequencing to characterize LBN-induced transcriptional changes in the medial preoptic area (mPOA), which underlies male reproductive behaviors. LBN produced sex-specific alterations in gene expression, with many transcripts showing changes in opposite directions. Numerous transcripts altered by LBN in males are regulated by estradiol, linking hormonal changes to molecular changes in the mPOA. Pathway analysis revealed that LBN induced changes in neurosignaling and immune signaling in males and females, respectively. Collectively, these studies reveal novel neurobiological mechanisms by which early life adversity can alter reproductive strategies.

Transcriptomics in the nucleus accumbens shell reveal sex- and reinforcer-specific signatures associated with morphine and sucrose craving.

Incubation of craving is a well-documented phenomenon referring to the intensification of drug craving over extended abstinence. The neural adaptations that occur during forced abstinence following chronic drug taking have been a topic of intense study. However, little is known about the transcriptomic changes occurring throughout this window of time. To define gene expression changes associated with morphine consumption and extended abstinence, male and female rats underwent 10 days of morphine self-administration. Separate drug-naive rats self-administered sucrose in order to compare opioid-induced changes from those associated with natural, non-drug rewards. After one or 30 days of forced abstinence, rats were tested for craving, or nucleus accumbens shell tissue was dissected for RNA sequencing. Morphine consumption was predictive of drug seeking after extended (30 days) but not brief (1 day) abstinence in both sexes. Extended abstinence was also associated with robust sex- and reinforcer-specific changes in gene expression, suggesting sex differences underlying incubation of morphine and sucrose seeking respectively. Importantly, these changes in gene expression occurred without re-exposure to drug-paired cues, indicating that chronic morphine causes long-lasting changes in gene expression that prime the system for increased craving. These findings lay the groundwork for identifying specific therapeutic targets for curbing opioid craving without impacting the natural reward system in males and females.

Chronic paternal morphine exposure increases sensitivity to morphine-derived pain relief in male progeny.

Parental history of opioid exposure is seldom considered when prescribing opioids for pain relief. To explore whether parental opioid exposure may affect sensitivity to morphine in offspring, we developed a "rat pain scale" with high-speed imaging, machine learning, and mathematical modeling in a multigenerational model of paternal morphine self-administration. We find that the most commonly used tool to measure mechanical sensitivity in rodents, the von Frey hair, is not painful in rats during baseline conditions. We also find that male progeny of morphine-treated sires had no baseline changes in mechanical pain sensitivity but were more sensitive to the pain-relieving effects of morphine. Using RNA sequencing across pain-relevant brain regions, we identify gene expression changes within the regulator of G protein signaling family of proteins that may underlie this multigenerational phenotype. Together, this rat pain scale revealed that paternal opioid exposure increases sensitivity to morphine's pain-relieving effects in male offspring.

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.

Early life adversity promotes resilience to opioid addiction-related phenotypes in male rats and sex-specific transcriptional changes.

Experiencing some early life adversity can have an "inoculating" effect that promotes resilience in adulthood. However, the mechanisms underlying stress inoculation are unknown, and animal models are lacking. Here we used the limited bedding and nesting (LBN) model of adversity to evaluate stress inoculation of addiction-related phenotypes. In LBN, pups from postnatal days 2 to 9 and their dams were exposed to a low-resource environment. In adulthood, they were tested for addiction-like phenotypes and compared to rats raised in standard housing conditions. High levels of impulsivity are associated with substance abuse, but in males, LBN reduced impulsive choice compared to controls. LBN males also self-administered less morphine and had a lower breakpoint on a progressive ratio reinforcement schedule than controls. These effects of LBN on addiction-related behaviors were not found in females. Because the nucleus accumbens (NAc) mediates these behaviors, we tested whether LBN altered NAc physiology in drug-naïve and morphine-exposed rats. LBN reduced the frequency of spontaneous excitatory postsynaptic currents in males, but a similar effect was not observed in females. Only in males did LBN prevent a morphine-induced increase in the AMPA/NMDA ratio. RNA sequencing was performed to delineate the molecular signature in the NAc associated with LBN-derived phenotypes. LBN produced sex-specific changes in transcription, including in genes related to glutamate transmission. Collectively, these studies reveal that LBN causes a male-specific stress inoculation effect against addiction-related phenotypes. Identifying factors that promote resilience to addiction may reveal novel treatment options for patients.

Cyclic AMP response element-binding protein is required in excitatory neurons in the forebrain to sustain wakefulness.

The molecular and intracellular signaling processes that control sleep and wake states remain largely unknown. A consistent observation is that the cyclic adenosine monophosphate (AMP) response element-binding protein (CREB), an activity-dependent transcription factor, is differentially activated during sleep and wakefulness. CREB is phosphorylated by the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway as well as other kinases, and phosphorylated CREB promotes the transcription of target genes. Genetic studies in flies and mice suggest that CREB signaling influences sleep/wake states by promoting and stabilizing wakefulness. However, it remains unclear where in the brain CREB is required to drive wakefulness. In rats, CREB phosphorylation increases in the cerebral cortex during wakefulness and decreases during sleep, but it is not known if this change is functionally relevant to the maintenance of wakefulness. Here, we used the Cre/lox system to conditionally delete CREB in the forebrain (FB) and in the locus coeruleus (LC), two regions known to be important for the production of arousal and wakefulness. We used polysomnography to measure sleep/wake levels and sleep architecture in conditional CREB mutant mice and control littermates. We found that FB-specific deletion of CREB decreased wakefulness and increased non-rapid eye movement sleep. Mice lacking CREB in the FB were unable to sustain normal periods of wakefulness. On the other hand, deletion of CREB from LC neurons did not change sleep/wake levels or sleep/wake architecture. Taken together, these results suggest that CREB is required in neurons within the FB but not in the LC to promote and stabilize wakefulness.

Consequences of Parental Opioid Exposure on Neurophysiology, Behavior, and Health in the Next Generations.

Substance abuse and the ongoing opioid epidemic represents a large societal burden. This review will consider the long-term impact of opioid exposure on future generations. Prenatal, perinatal, and preconception exposure are reviewed with discussion of both maternal and paternal influences. Opioid exposure can have long-lasting effects on reproductive function, gametogenesis, and germline epigenetic programming, which can influence embryogenesis and alter the developmental trajectory of progeny. The potential mechanisms by which preconception maternal and paternal opioid exposure produce deleterious consequences on the health, behavior, and physiology of offspring that have been identified by clinical and animal studies will be discussed. The timing, nature, dosing, and duration of prenatal opioid exposure combined with other important environmental considerations influence the extent to which these manipulations affect parents and their progeny. Epigenetic inheritance refers to the transmission of environmental insults across generations via mechanisms independent of the DNA sequence. This topic will be further explored in the context of prenatal, perinatal, and preconception opioid exposure for both the maternal and paternal lineage.

Aging reduces the sensitivity to the reinforcing efficacy of morphine.

The US geriatric population is growing and using more opioids than ever before. The purpose of this study was to determine whether aging influenced the reinforcing efficacy of morphine in male and female rats using a rodent intravenous self-administration paradigm. Male and female aged (20-24 months) and young (2-4 months) Wistar rats were tested at 2 doses of morphine (0.75 mg/kg/infusion and 0.25 mg/kg/infusion). During 10 days of self-administration, aged rats took significantly less morphine than their younger counterparts at the 0.25 mg/kg/infusion dose. Aged males also earned significantly fewer infusions on a progressive ration reinforcement schedule at this dose, suggesting that the reinforcing efficacy of morphine is decreased for this group at this dose. These effects dissipated when a separate group of animals had access to the 0.75 mg/kg/infusion dose for both sexes. Our results indicate that morphine is less reinforcing at lower doses in aged male, but not female rats. This research has potential clinical implications for the chronic treatments involving opioids in aged individuals.