Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

Distinct engrams control fear and extinction memory.

Memories are stored in engram cells, which are necessary and sufficient for memory recall. Recalling a memory might undergo reconsolidation or extinction. It has been suggested that the original memory engram is reactivated during reconsolidation so that memory can be updated. Conversely, during extinction training, a new memory is formed that suppresses the original engram. Nonetheless, it is unknown whether extinction creates a new engram or modifies the original fear engram. In this study, we utilized the Daun02 procedure, which uses c-Fos-lacZ rats to induce apoptosis of strongly activated neurons and examine whether a new memory trace emerges as a result of a short or long reactivation, or if these processes rely on modifications within the original engram located in the basolateral amygdala (BLA) and infralimbic (IL) cortex. By eliminating neurons activated during consolidation and reactivation, we observed significant impacts on fear memory, highlighting the importance of the BLA engram in these processes. Although we were unable to show any impact when removing the neurons activated after the test of a previously extinguished memory in the BLA, disrupting the IL extinction engram reactivated the aversive memory that was suppressed by the extinction memory. Thus, we demonstrated that the IL cortex plays a crucial role in the network involved in extinction, and disrupting this specific node alone is sufficient to impair extinction behavior. Additionally, our findings indicate that extinction memories rely on the formation of a new memory, supporting the theory that extinction memories rely on the formation of a new memory, whereas the reconsolidation process reactivates the same original memory trace.

Progestin-primed ovarian stimulation.

PURPOSE OF REVIEW: The use of progestins as pituitary suppressors has increased progressively, along with more detailed indications for their use, thereby consolidating an alternative approach to the personalization of ovarian stimulation. RECENT FINDINGS: Based on the ability of progesterone to inhibit ovulation, progestins have been used in ovarian stimulation (OS) follicular protocols to prevent a luteinizing hormone surge in patients undergoing in vitro fertilization (IVF), as an alternative to gonadotropin-releasing hormone (GnRH) analogue administration. This review explores the different types of progestogen protocols and their efficacy depending on the type of population or reproductive procedure in which they are administered and in comparison with that of GnRH analogues. Their effect on oocytes and embryos and their safety and cost-effectiveness are also analyzed. SUMMARY: Progestins have proven their effectiveness as a gonadotropin adjuvant in terms of ovarian response, reproductive outcome, and safety. In addition, they offer the convenience of oral administration and a lower cost than GnRH analogues. Whereas oocytes or embryos should be vitrified as it displaces the receptive period with the consequent asynchrony between embryo and endometrium. The evidence endorses progestins as a more friendly approach to OS, especially when frozen-thawed embryo transfer is planned.

l-Arginine supplementation for pregnant and lactating sows may improve the performance of piglets: A systematic review.

The objective was to conduct a systematic review to clarify the effects of l-arginine supplementation in pregnant and lactating sows on plasma hormone levels, milk production and composition, the body condition of sows and piglet performance. In April 2023, an online search and a systematic search were performed in the following databases: Embase, Scopus, SciELO, Web of Science, PubMed and Science Direct. The combinations of keywords used were sow and arginine and lactation; sow and arginine and lactating; sow and arginine and gestation; sow and arginine and gestating; sow and arginine and pregnancy; sow and arginine and reproduction; piglet and arginine; and sow and arginine and mammary gland. In total, 21 scientific articles with original data were selected according to preestablished criteria. Among the 21 articles, seven (33%) reported measurements of some plasma hormones, and among these, six reported an increase in the levels of at least one hormone, namely, estradiol, insulin-like growth factor 1 (IGF-1), insulin, follicle stimulating hormone, growth hormone or prolactin, with l-arginine supplementation. The parameters of milk were evaluated in 11 studies (52%), one reported an increase in protein content, and one reported an increase in IGF-1 content in milk with supplementation of this amino acid. Of the 14 studies that evaluated the performance parameters of piglets, only four reported improvements in some parameters of piglets from sows that received supplementation. Dietary supplementation of arginine for sows in the final third of gestation and/or lactation may alter the plasma levels of some hormones, which may reflect in greater development of the mammary gland tissue and, consequently, promote benefits on the performance of piglets. However, more studies are needed to evaluate the real impact of this amino acid supplementation on the physiology of the sows, in general, and the performance of suckling piglets.

Novel Constraints on Axions Produced in Pulsar Polar-Cap Cascades.

Axions can be copiously produced in localized regions of neutron star magnetospheres where the ambient plasma is unable to efficiently screen the induced electric field. As these axions stream away from the neutron star they can resonantly transition into photons, generating a large broadband contribution to the neutron star's intrinsic radio flux. In this Letter, we develop a comprehensive end-to-end framework to model this process from the initial production of axions to the final detection of radio photons, and derive constraints on the axion-photon coupling, g_{aγγ}, using observations of 27 nearby pulsars. We study the modeling uncertainty in the sourced axion spectrum by comparing predictions from 2.5 dimensional particle-in-cell simulations with those derived using a semianalytic model; these results show remarkable agreement, leading to constraints on the axion-photon coupling that typically differ by a factor of no more than ∼2. The limits presented here are the strongest to date for axion masses 10^{-8} eV≲m_{a}≲10^{-5} eV, and crucially do not rely on the assumption that axions are dark matter.

Similar reproductive outcomes between lesbian-shared IVF (ROPA) and IVF with autologous oocytes.

PURPOSE: To compare reproductive outcomes of the ROPA method (reception of oocytes from partner) to IVF with autologous oocytes. To study the impact of the absence of a genetic link between the embryo and its recipient in reproductive outcomes. METHODS: Retrospective multicentric cohort study performed from January 2011 to December 2020 in 18 fertility clinics in Spain. A total of 99 ROPA (73 couples) and 2929 non-ROPA cycles (2334 couples or single patients) of women younger than 38 years old with no known female fertility disorder were included. Clinical outcomes were compared between both groups and included positive pregnancy test, clinical pregnancy, miscarriage, ectopic pregnancy, pre-term birth, live birth, weeks of gestation at birth, and newborn weight at birth. RESULTS: No differences were found between groups in clinical outcomes. The total clinical pregnancy rates per embryo transfer were 57% and 50.2% (p = 0.15) and the live-birth rates were 46.1% and 40.9% (p = 0.14) for the ROPA and non-ROPA groups, respectively. When adjusted to age and BMI of donors and recipients, there were also no differences in live-birth rates between both groups. The cumulative live-birth rate per ROPA cycle was 73.7% and the cumulative live-birth rate per couple was 78.3%. CONCLUSION: Clinical outcomes following the ROPA method and IVF with autologous oocytes were found to be similar. These findings suggest no impact of the absence of genetic ties between the embryo and the uterus on reproductive treatments' outcomes. Data regarding the outcomes of the ROPA method are reassuring.

Impact of low serum progesterone levels on the day of embryo transfer on pregnancy outcome: a prospective cohort study in artificial cycles with vaginal progesterone.

STUDY QUESTION: Is there a serum progesterone (P) threshold on the day of embryo transfer (ET) in artificial endometrium preparation cycles below which the chances of ongoing pregnancy are reduced? SUMMARY ANSWER: Serum P levels <8.8 ng/ml on the day of ET lower ongoing pregnancy rate (OPR) in both own or donated oocyte cycles. WHAT IS KNOWN ALREADY: We previously found that serum P levels <9.2 ng/ml on the day of ET significantly decrease OPR in a sample of 211 oocyte donation recipients. Here, we assessed whether these results are applicable to all infertile patients under an artificial endometrial preparation cycle, regardless of the oocyte origin. STUDY DESIGN, SIZE, DURATION: This prospective cohort study was performed between September 2017 and November 2018 and enrolled 1205 patients scheduled for ET after an artificial endometrial preparation cycle with estradiol valerate and micronized vaginal P (MVP, 400 mg twice daily). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients ≤50 years old with a triple-layer endometrium ≥6.5 mm underwent transfer of one or two blastocysts. A total of 1150 patients treated with own oocytes without preimplantation genetic testing for aneuploidies (PGT-A) (n = 184), own oocytes with PGT-A (n = 308) or donated oocytes (n = 658) were analyzed. The primary endpoint was the OPR beyond pregnancy week 12 based on serum P levels measured immediately before ET. MAIN RESULTS AND THE ROLE OF CHANCE: Women with serum P levels <8.8 ng/ml (30th percentile) had a significantly lower OPR (36.6% vs 54.4%) and live birth rate (35.5% vs 52.0%) than the rest of the patients. Multivariate logistic regression showed that serum P < 8.8 ng/ml was an independent factor influencing OPR in the overall population and in the three treatment groups. A significant negative correlation was observed between serum P levels and BMI, weight and time between the last P dose and blood tests and a positive correlation was found with age, height and number of days on HRT. Multivariate logistic regression showed that only body weight was an independent factor for presenting serum P levels <8.8 ng/ml. Obstetrical and perinatal outcomes did not differ in patients with ongoing pregnancy regardless of serum P levels being above/below 8.8 ng/ml. LIMITATIONS, REASONS FOR CAUTION: Only women with MVP were included. Extrapolation to other P administration forms needs to be validated. WIDER IMPLICATIONS OF THE FINDINGS: This study identified the threshold of serum P as 8.8 ng/ml on the day of ET for artificial endometrial preparation cycles necessary to optimize outcomes, in cycles with own or donated oocytes. One-third of patients receiving MVP show inadequate levels of serum P that, in turn, impact the success of the ART cycle. Monitoring P levels in the mid-luteal phase is recommended when using MVP to adjust the doses according to the needs of the patient. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NCT03272412.

Relationship between beta-adrenergic agonists, calpain system activity and beef texture: A systematic review.

The objective was to conduct a systematic review to evaluate the effects of dietary supplementation with beta-adrenergic agonists on calpains and calpastatin activity in bovine muscle and changes in meat tenderness. A survey was conducted in June 2019 on Science Direct, Web of Science, Scopus, PubMed and Capes Periodicals, using four keyword combinations: agonist and calpain and cattle; agonist and calpain and bovine; agonist and calpain and heifers; agonist and calpain and steers. Thirteen studies were selected, 54% concluded that supplementation with beta-adrenergic agonists increases calpastatin activity, 23% observed increase in their gene expression and 23% reported no effect on activity or expression of this enzyme. Nine studies evaluated the influence of beta-adrenergic agonists supplementation on meat texture and all found an increase in shear force values. There is strong evidence that beta-adrenergic agonists may increase calpastatin activity in the muscle, causing damage to meat tenderness.

Discovery of Potent, Reversible, and Competitive Cruzain Inhibitors with Trypanocidal Activity: A Structure-Based Drug Design Approach.

A virtual screening conducted with nearly 4 000 000 compounds from lead-like and fragment-like subsets enabled the identification of a small-molecule inhibitor (1) of the Trypanosoma cruzi cruzain enzyme, a validated drug target for Chagas disease. Subsequent comprehensive structure-based drug design and structure-activity relationship studies led to the discovery of carbamoyl imidazoles as potent, reversible, and competitive cruzain inhibitors. The most potent carbamoyl imidazole inhibitor (45) exhibited high affinity with a Ki value of 20 nM, presenting both in vitro and in vivo activity against T. cruzi. Furthermore, the most promising compounds reduced parasite burden in vivo and showed no toxicity at a dose of 100 mg/kg. These carbamoyl imidazoles are structurally attractive, nonpeptidic, and easy to prepare and synthetically modify. Finally, these results further advance our understanding of the noncovalent mode of inhibition of this pharmaceutically relevant enzyme, building strong foundations for drug discovery efforts.

Antiplasmodial profile of selected compounds from Malaria Box: in vitro evaluation, speed of action and drug combination studies.

BACKGROUND: Artemisinin-based combination therapy (ACT) is used as the first-line treatment of uncomplicated malaria caused by the Plasmodium falciparum parasite and chloroquine-resistant Plasmodium vivax parasites. Evidence of resistance to ACT has been reported in Cambodia, and without new and effective anti-malarial agents, malaria burden and mortality will rise. METHODS: The used MolPrint 2D fingerprints and the Tanimoto similarity index were used to perform a structural similarity search within the Malaria Box collection to select diverse molecular scaffolds that are different from artesunate. Next, the inhibitory potency against the P. falciparum 3D7 strain (SYBR Green I inhibition assay) and the cytotoxicity against HepG2 cells (MTT and neutral red assays) were evaluated. Then, the speed of action, the combination profile of selected inhibitors with artesunate, and the P. berghei in vivo activity of the best compounds were assessed. RESULTS: A set of 11 structurally diverse compounds from the Malaria Box with a similarity threshold of less than 0.05 was selected and compared with artesunate. The in vitro inhibitory activity of each compound confirmed the reported potencies (IC50 values ranging from 0.005 to 1 µM). The cytotoxicity of each selected compound was evaluated and used to calculate the selectivity index (SI values ranging from 15.1 to 6100). Next, both the speed of action and the combination profile of each compound with artesunate was assessed. Acridine, thiazolopyrimidine, quinoxaline, benzimidazole, thiophene, benzodiazepine, isoxazole and pyrimidoindole derivatives showed fast in vitro inhibitory activity of parasite growth, whereas hydrazinobenzimidazole, indenopyridazinone and naphthalenone derivatives were slow-acting in vitro inhibitors. Combinatory profile evaluation indicated that thiazolopyrimidinone and benzodiazepine derivatives have an additive profile, suggesting that the combination of these inhibitors with artesunate is favourable for in vitro inhibitory activity. The remaining compounds showed an antagonistic combinatory profile with artesunate. The collected data indicated that the indenopyridazinone derivative, a bc1 complex inhibitor, had a similar association profile in combination with proguanil when compared to atovaquone combined with proguanil, thereby corroborating the correlation between the molecular target and the combination profile. Lastly, the in vivo activity of the thiazolopyrimidinone and benzodiazepine derivatives were assessed. Both compounds showed oral efficacy at 50 mg/kg in a mouse model of Plasmodium berghei malaria (64% and 40% reduction in parasitaemia on day 5 post-infection, respectively). CONCLUSIONS: The findings in this paper shed light on the relationship among the speed of action, molecular target and combinatory profile and identified new hits with in vivo activity as candidates for anti-malarial combination therapy.

Prolonged Exposure to Alcohol Vapor Causes Change in Cardiovascular Function in Female but not in Male Rats.

BACKGROUND: Alcohol abuse is a health concern worldwide. Studies have associated alcohol abuse with cardiovascular impairments. In this study, we investigated differences in the effects of chronic alcohol vapor exposure on cardiovascular function between male and female rats by using the alcohol vapor chamber method to induce alcohol addiction-like behaviors in rats. METHODS: We exposed male and female Long-Evans rats to alcohol vapor for 14 hours, followed by ethanol withdrawal for 10 hours, for 30 consecutive days or room air (control groups). The animals underwent preparation for the surgical implantation of cannulas into femoral vessels, for allowing the assessment of the basal arterial pressure and heart rate values, baroreflex function, and autonomic activity. RESULTS: Female control rats showed higher basal heart rate compared to male control rats. Chronic alcohol vapor inhalation reduced basal heart rate in females, but not in males; this effect was followed by an increase in the parasympathetic tone of the heart. Further, female rats subjected to alcohol vapor showed an increase in the baroreflex activity. CONCLUSIONS: These findings suggest that females are more sensitive to chronic alcohol vapor exposure than males because they had a reduction in basal heart rate and changes in the baroreflex activity.

New technologies for examining the role of neuronal ensembles in drug addiction and fear.

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. In addition, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches--Daun02 inactivation, FACS sorting of activated neurons and Fos-GFP transgenic rats--that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools--Fos-tTA transgenic mice and inactivation of CREB-overexpressing neurons--that have been used to study the role of neuronal ensembles in conditioned fear.

Distinct Fos-Expressing Neuronal Ensembles in the Ventromedial Prefrontal Cortex Mediate Food Reward and Extinction Memories.

UNLABELLED: In operant learning, initial reward-associated memories are thought to be distinct from subsequent extinction-associated memories. Memories formed during operant learning are thought to be stored in "neuronal ensembles." Thus, we hypothesize that different neuronal ensembles encode reward- and extinction-associated memories. Here, we examined prefrontal cortex neuronal ensembles involved in the recall of reward and extinction memories of food self-administration. We first trained rats to lever press for palatable food pellets for 7 d (1 h/d) and then exposed them to 0, 2, or 7 daily extinction sessions in which lever presses were not reinforced. Twenty-four hours after the last training or extinction session, we exposed the rats to either a short 15 min extinction test session or left them in their homecage (a control condition). We found maximal Fos (a neuronal activity marker) immunoreactivity in the ventral medial prefrontal cortex of rats that previously received 2 extinction sessions, suggesting that neuronal ensembles in this area encode extinction memories. We then used the Daun02 inactivation procedure to selectively disrupt ventral medial prefrontal cortex neuronal ensembles that were activated during the 15 min extinction session following 0 (no extinction) or 2 prior extinction sessions to determine the effects of inactivating the putative food reward and extinction ensembles, respectively, on subsequent nonreinforced food seeking 2 d later. Inactivation of the food reward ensembles decreased food seeking, whereas inactivation of the extinction ensembles increased food seeking. Our results indicate that distinct neuronal ensembles encoding operant reward and extinction memories intermingle within the same cortical area. SIGNIFICANCE STATEMENT: A current popular hypothesis is that neuronal ensembles in different prefrontal cortex areas control reward-associated versus extinction-associated memories: the dorsal medial prefrontal cortex (mPFC) promotes reward seeking, whereas the ventral mPFC inhibits reward seeking. In this paper, we use the Daun02 chemogenetic inactivation procedure to demonstrate that Fos-expressing neuronal ensembles mediating both food reward and extinction memories intermingle within the same ventral mPFC area.

Chronic nicotine activates stress/reward-related brain regions and facilitates the transition to compulsive alcohol drinking.

Alcohol and nicotine are the two most co-abused drugs in the world. Previous studies have shown that nicotine can increase alcohol drinking in nondependent rats, yet it is unknown whether nicotine facilitates the transition to alcohol dependence. We tested the hypothesis that chronic nicotine will speed up the escalation of alcohol drinking in rats and that this effect will be accompanied by activation of sparsely distributed neurons (neuronal ensembles) throughout the brain that are specifically recruited by the combination of nicotine and alcohol. Rats were trained to respond for alcohol and made dependent using chronic, intermittent exposure to alcohol vapor, while receiving daily nicotine (0.8 mg/kg) injections. Identification of neuronal ensembles was performed after the last operant session, using immunohistochemistry. Nicotine produced an early escalation of alcohol drinking associated with compulsive alcohol drinking in dependent, but not in nondependent rats (air exposed), as measured by increased progressive-ratio responding and increased responding despite adverse consequences. The combination of nicotine and alcohol produced the recruitment of discrete and phenotype-specific neuronal ensembles (∼4-13% of total neuronal population) in the nucleus accumbens core, dorsomedial prefrontal cortex, central nucleus of the amygdala, bed nucleus of stria terminalis, and posterior ventral tegmental area. Blockade of nicotinic receptors using mecamylamine (1 mg/kg) prevented both the behavioral and neuronal effects of nicotine in dependent rats. These results demonstrate that nicotine and activation of nicotinic receptors are critical factors in the development of alcohol dependence through the dysregulation of a set of interconnected neuronal ensembles throughout the brain.

Context-induced reinstatement of methamphetamine seeking is associated with unique molecular alterations in Fos-expressing dorsolateral striatum neurons.

Context-induced reinstatement of drug seeking is a well established animal model for assessing the neural mechanisms underlying context-induced drug relapse, a major factor in human drug addiction. Neural activity in striatum has previously been shown to contribute to context-induced reinstatement of heroin, cocaine, and alcohol seeking, but not yet for methamphetamine seeking. In this study, we found that context-induced reinstatement of methamphetamine seeking increased expression of the neural activity marker Fos in dorsal but not ventral striatum. Reversible inactivation of neural activity in dorsolateral but not dorsomedial striatum using the GABA agonists muscimol and baclofen decreased context-induced reinstatement. Based on our previous findings that Fos-expressing neurons play a critical role in conditioned drug effects, we assessed whether context-induced reinstatement was associated with molecular alterations selectively induced within context-activated Fos-expressing neurons. We used fluorescence-activated cell sorting to isolate reinstatement-activated Fos-positive neurons from Fos-negative neurons in dorsal striatum and used quantitative PCR to assess gene expression within these two populations of neurons. Context-induced reinstatement was associated with increased expression of the immediate early genes Fos and FosB and the NMDA receptor subunit gene Grin2a in only Fos-positive neurons. RNAscope in situ hybridization confirmed that Grin2a, as well as Grin2b, expression were increased in only Fos-positive neurons from dorsolateral, but not dorsomedial, striatum. Our results demonstrate an important role of dorsolateral striatum in context-induced reinstatement of methamphetamine seeking and that this reinstatement is associated with unique gene alterations in Fos-expressing neurons.

Role of nucleus accumbens shell neuronal ensembles in context-induced reinstatement of cocaine-seeking.

Environmental contexts previously associated with drug use provoke relapse to drug use in humans and reinstatement of drug seeking in animal models of drug relapse. We examined whether context-induced reinstatement of cocaine seeking is mediated by activation of context-selected nucleus accumbens neurons. We trained rats to self-administer cocaine in Context A and extinguished their lever-pressing in a distinct Context B. On test day, reexposure to the cocaine-associated Context A reinstated cocaine seeking and increased expression of the neural activity marker Fos in 3.3% of accumbens shell and 1.6% of accumbens core neurons. To assess a causal role for these activated neurons, we used the Daun02 inactivation procedure to selectively inactivate these neurons. We trained c-fos-lacZ transgenic rats to self-administer cocaine in Context A and extinguished their lever-pressing in Context B. On induction day, we exposed rats to either Context A or a novel Context C for 30 min and injected Daun02 or vehicle into accumbens shell or core 60 min later. On test day, 3 d after induction day, the ability of Context A to reinstate cocaine seeking and increase neuronal activity in accumbens shell was attenuated when Daun02 was previously injected after exposure to Context A. Daun02 injections after exposure to the novel Context C had no effect on context-induced reinstatement of cocaine seeking despite much greater numbers of Fos-expressing neurons induced by Context C. Daun02 injections in accumbens core had no effect. Our data suggest that context-induced reinstatement of cocaine seeking is mediated by activation of context-selected accumbens shell but not core neuronal ensembles.

Stress vulnerability during adolescence: comparison of chronic stressors in adolescent and adult rats.

OBJECTIVE: This study investigated the physiological and somatic changes evoked by daily exposure to the same type of stressor (homotypic) or different aversive stressor stimuli (heterotypic) in adolescent and adult rats, with a focus on cardiovascular function. The long-term effects of stress exposure during adolescence were also investigated longitudinally. METHODS: Male Wistar rats were exposed to repeated restraint stress (RRS, homotypic) or chronic variable stress (CVS, heterotypic). RESULTS: Adrenal hypertrophy, thymus involution, and elevated plasma glucocorticoid were observed only in adolescent animals, whereas reduction in body weight was caused by both stress regimens in adults. CVS increased mean arterial pressure (adolescent: p = .001; adult: p = .005) and heart rate (HR; adolescent: p = .020; adult: p = .011) regardless of the age, whereas RRS increased blood pressure selectively in adults (p = .001). Rest tachycardia evoked by CVS was associated with increased cardiac sympathetic activity in adults, whereas a decreased cardiac parasympathetic activity was observed in adolescent animals. Changes in cardiovascular function and cardiac autonomic activity evoked by both CVS and RRS were followed by alterations in baroreflex activity and vascular reactivity to vasoconstrictor and vasodilator agents in adolescent adult animals. Except for the circulating glucocorticoid change, all alterations observed during adolescence were reversed in adulthood. CONCLUSIONS: These findings suggest a stress vulnerability of adolescents to somatic and neuroendocrine effects regardless of stress regimen. Our results indicated an age-stress type-specific influence in stress-evoked cardiovascular/autonomic changes. Data suggest minimal consequences in adulthood of stress during adolescence.

Live birth after embryo transfer in an unresponsive thin endometrium.

A healthy term livebirth in a 35-year-old woman with ovarian failure, hypoplastic uterus and atrophic endometrium after cancer treatment with chemotherapy and radiotherapy was achieved by ovum donation. The detection of a receptive endometrium using a new diagnostic tool of endometrial receptivity encouraged the medical team to carry on with this poor prognosis case. To the best of our knowledge, here we report the thinnest endometrium to date in which a healthy, full term live birth has been described in assisted conception.

Long-lasting mechanical hypersensitivity and CRF receptor type-1 neuron activation in the BNST following adolescent ethanol exposure.

BACKGROUND: Adolescent alcohol use can produce long-lasting alterations in brain function, potentially leading to adverse health outcomes in adulthood. Emerging evidence suggests that chronic alcohol use can increase pain sensitivity or exacerbate existing pain conditions, but the potential neural mechanisms underlying these effects require further investigation. Here, we evaluate the impact of chronic ethanol vapor on mechanical sensitivity over the course of acute and protracted withdrawal in adolescent and adult male and female mice, and its potential association with alterations in corticotropin-releasing factor (CRF) signaling within the bed nucleus of the stria terminalis (BNST). METHODS: Adolescent and adult male and female mice underwent intermittent ethanol vapor exposure on 4 days/week for 2 weeks. Mechanical thresholds were evaluated 5 h and 7, 14, 21, and 28 d after cessation of ethanol exposure using the von Frey test. For mice with a history of adolescent ethanol exposure, brains were collected for in situ RNAscope processing after the final test. Messenger RNA expression of c-Fos, Crfr1, and Crf in the BNST subregions was examined. RESULTS: Exposure to intermittent ethanol vapor induced persistent mechanical hypersensitivity during withdrawal in both adolescent and adult mice. Notably, the effect was more transient in mice exposed to ethanol during adulthood, resolving by day 28 after ethanol exposure. Furthermore, both male and female mice with a history of adolescent ethanol exposure exhibited increased activation of CRF receptor type 1 (CRFR1) neurons within the dorsolateral BNST. CONCLUSIONS: Our results support the conclusion that intermittent ethanol exposure can induce mechanical hypersensitivity, potentially through the activation of BNST CRFR1 neurons. These findings provide a basis for future studies aimed at evaluating specific subpopulations of BNST neurons and their contribution to pain in individuals with a history of alcohol use.

BNST CRF receptor type 1 modulates mechanical hypersensitivity induced by adolescent alcohol exposure in adult female mice.

RATIONALE: Alcohol exposure during adolescence has been linked to long-lasting behavioral consequences, contributing to the development of alcohol use disorder. Negative affect and chronic pain during alcohol withdrawal are critical factors influencing problematic alcohol use and relapse. Our previous research demonstrated that adolescent intermittent ethanol (AIE) vapor exposure elicits sex-specific negative affect-like behavior in adult mice following stress exposure. Additionally, AIE induces persistent mechanical hypersensitivity, which is accompanied by increased activation of corticotropin-releasing factor receptor type 1 (CRFR1) neurons in the dorsolateral bed nucleus of the stria terminalis (dlBNST). OBJECTIVES: This study extends previous work by investigating plasma corticosterone levels and CRFR1 protein expression in the dlBNST following restraint stress exposure in adult mice with an AIE history. We also aim to explore the role of dlBNST CRFR1 signaling in mediating negative affect-like behavior and mechanical hypersensitivity. RESULTS: Female mice exhibited elevated plasma corticosterone levels compared to males following restraint stress. Moreover, females with AIE history showed higher expression of CRFR1 protein in the dlBNST compared to air controls. Antagonism of CRFR1 in the dlBNST blocked AIE-induced mechanical hypersensitivity in adult females but did not affect stress-induced negative affect-like behavior. In alcohol-naïve females, intra-dlBNST administration of a CRFR1 agonist induced mechanical hypersensitivity. CONCLUSIONS: These findings provide new insights into the neurobiological mechanisms underlying stress-induced negative affect and pain-related behavior, both influenced by a history of adolescent alcohol exposure. The results suggest that CRFR1 antagonists warrant further investigation for their potential in addressing alcohol-related chronic pain.