Autorepression of yeast Hsp70 cochaperones by intramolecular interactions involving their J-domains.

The 70 kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s to trigger ATP hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1's ATPase, indicate the presence of yet uncharacterized intramolecular dynamic interactions between the J-domains and the remaining C-terminal segments of these proteins. Taken together, the data suggest an autoregulatory role to these intramolecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol.

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.

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins.

The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

Design of an Arabidopsis thaliana reporter line to detect heat-sensing and signaling mutants.

BACKGROUND: Global warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a heat-responsive Arabidopsis thaliana reporter line that allows an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature. METHODS: A transgenic Arabidopsis thaliana reporter line named "Heat-Inducible Bioluminescence And Toxicity" (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and D-amino acid oxidase, whose expression is toxic in the presence of D-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of D-valine and analyzed for survival rate, bioluminescence and HSP gene expression. RESULTS: Whereas at 22 °C, HIBAT seedlings grew unaffected by D-valine, and all survived iterative heat treatments without D-valine, 98% died following heat treatments on D-valine. The HSP17.3B promoter was highly specific to heat as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. RNAseq analysis of heat-treated HIBAT seedlings showed a strong correlation with expression profiles of two wild type lines, confirming that HIBAT does not significantly differ from its Col-0 parent. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants, apparently defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at non-heat-shock temperatures. CONCLUSION: HIBAT is a valuable candidate tool to identify Arabidopsis mutants defective in the response to high temperature stress. It opens new avenues for future research on the regulation of HSP expression and for understanding the mechanisms of plant acquired thermotolerance.

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.

[Discordant Down syndrome risk calculation with low maternal serum markers: About five cases of digynic triploidies]. / Risque de trisomie 21 discordant en cas de marqueurs sériques très bas : à propos de cinq cas de triploïdie digynique.

OBJECTIVE: We compare the risk of Down syndrome among five patients carrying a foetus with digynic triploidy and suggest a course of action for these particular serological profiles. METHODS: The concentrations of the different markers used are transformed into multiples of the median by using each of the three software types present on the French market which then determine the risk of Down syndrome. RESULTS: For comparable biochemical and ultrasound profiles, the risk of Down syndrome turns out to be vastly different depending on the type of software employed. The relevance of an immediate diagnostic procedure, of a cell free DNA test or of a basic ultrasound follow-up then arises, leading to a potentially variable care pathway for the patient. CONCLUSIONS: This study confirms that for this type of biochemical profile, the laboratory's advisory service is fundamental, that a control ultrasound is essential and that an invasive procedure must be used almost invariably due to the extremely substantial risk factors.

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.

How do plants feel the heat and survive?

Climate change is increasingly affecting the quality of life of organisms on Earth. More frequent, extreme, and lengthy heat waves are contributing to the sixth mass extinction of complex life forms in the Earth's history. From an anthropocentric point of view, global warming is a major threat to human health because it also compromises crop yields and food security. Thus, achieving agricultural productivity under climate change calls for closer examination of the molecular mechanisms of heat-stress resistance in model and crop plants. This requires a better understanding of the mechanisms by which plant cells can sense rising temperatures and establish effective molecular defenses, such as molecular chaperones and thermoprotective metabolites, as reviewed here, to survive extreme diurnal variations in temperature and seasonal heat waves.

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.

How do humans and plants feel the heat?

The 2021 Nobel prize was awarded for the discovery of the animal thermosensory channel TRPV1. We highlight notable shared features with the higher plant thermosensory channel CNGC2/4. Both channels respond to temperature-induced changes in plasma membrane fluidity, leading to hyperphosphorylation of the HSF1 transcription factor via a specific heat-signaling cascade.

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.

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.

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.

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.

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.