Neuroplasticity-related genes correlate with individual differences in distinct phases of oxycodone self-administration in male rats.

Opioid use disorder (OUD) is a chronic condition associated with long-lasting molecular and behavioral changes. Animals with prolonged access to opioids develop behaviors similar to human OUD. Identifying associated molecular changes can provide insight to underpinnings that lead to or maintain OUD. In pilot studies, we identified several miRNA targets that are altered by the administration of oxycodone. We selected mir182 for follow up as it was recently shown to be dysregulated in plasma of men administered oxycodone. In addition, mir182 is increased in reward-related brain regions of male rats following exposure to various addictive substances. The present study utilizes a long-access oxycodone self-administration paradigm to examine changes in mir182 and its mRNA targets associated with neuroplasticity, which may be involved in the maintenance of OUD-like phenotype in rats. Male rats were trained to self-administer oxycodone (0.1 mg/kg/infusion, i. v.) for 6 h daily sessions for 12 days. Each animal had a yoked saline control that received matched saline infusions. Animals were then tested on a progressive ratio schedule to measure motivation to obtain a single infusion of oxycodone. Drug seeking was measured following 28 days of forced abstinence using a 90-min cued/test. RTqPCR was utilized to measure mir182 and mRNA targets related to neuroplasticity (wnt3, plppr4, pou3f3, tle4, cacna2d, and bdnf) from the nucleus accumbens. Data revealed that animals responded on a continuum for oxycodone. When divided into two groups termed high- and low responders, animals diverged during self-administration acquisition and maintained differences in behavior and gene expression throughout the study. mir182 was upregulated in the nucleus accumbens of both high and low responders and negatively correlated with tle4, which showed a strong negative correlation with reinstatement behavior. mRNA target levels were correlated with behaviors associated with increased severity of OUD behavior in male rats.

Preconception opioids interact with mouse strain to alter morphine withdrawal in the next generation.

RATIONALE: Transgenerational effects of preconception morphine exposure in female rats have been reported which suggest that epigenetic modifications triggered by female opioid exposure, even when that exposure ends several weeks prior to pregnancy, has significant ramifications for their future offspring. OBJECTIVE: The current study compares two mouse strains with well-established genetic variation in their response to mu opioid receptor agonists, C57BL/6J (BL6) and 129S1/svlmJ (129) to determine whether genetic background modifies the impact of preconception opioid exposure. METHODS: Adolescent females from both strains were injected daily with morphine for a total of 10 days using an increasing dosing regimen with controls receiving saline. Several weeks after their final injection, aged-matched BL6 and 129 morphine (Mor-F0) or saline (Sal-F0) females were mated with drug naïve males to generate Mor-F1 and Sal-F1 offspring, respectively. As adults, F1 mice were made morphine dependent using thrice daily morphine injections for 4 days. On day 5, mice were administered either saline or morphine followed 3 h later by naloxone. Behavioral and physiological signs of withdrawal were then measured. RESULTS: Regardless of strain or sex, morphine-dependent Mor-F1 mice had significantly lower levels of withdrawal-induced corticosterone but significantly higher glucose levels when compared to Sal-F1 controls. In contrast, both strain- and preconception opioid exposure effects on physical signs of morphine dependence were observed.

Effects of Maternal Separation on Effort-based Responding for Sucrose Are Associated with c-Fos Expression in the Nucleus Accumbens Core.

In both people and animals, exposure to adverse experiences early in life can alter neurodevelopment and lead to long-term behavioral effects, including effects on reward processing. In the current study, we use a well-validated rodent model of maternal neglect, maternal separation (MS), to investigate the impact of early life adversity on reward learning and motivation and identify associated modifications in cellular activation in reward-relevant areas. Litters of Long-Evans rats were separated from the dam for either 15 min (brief) or 180 min (prolonged)/day from postnatal day (PND)2 to PND14. As adults, offspring were trained to lever press for a sucrose pellet using fixed ratio (FR) schedules and motivation was tested using a progressive ratio (PR) schedule over 10 daily sessions to assess sustained effects on effort-based responding. Immunohistochemical staining for c-Fos was conducted in a subset of animals that underwent an additional PR session. While there were no effects on reward learning, both MS180 males and females demonstrated increased effort-based responding on the first day of PR testing, while only MS180 males demonstrated a sustained increase in effort across all 10 days. MS180-induced changes in c-Fos expression in the dorsal and ventral striatum were observed, with subregion-specific effects along the rostrocaudal axis. Moreover, regression analyses suggest that motivated responding for a sucrose food reward in MS180-exposed, but not MS15-exposed animals, was associated with increased c-Fos expression in the rostral nucleus accumbens core. These findings implicate specific striatal regions in sex-specific modulation of sustained effort-based reward behavior following early life adversity.

Intergenerational effects of preconception opioids on glucose homeostasis and hepatic transcription in adult male rats.

Adolescence represents a period of significant neurodevelopment during which adverse experiences can lead to prolonged effects on disease vulnerability, including effects that can impact future offspring. Adolescence is a common period for the initiation of drug use, including the use of opioids. Beyond effects on central reward, opioids also impact glucose metabolism, which can impact the risk of diabetes. Moreover, recent animal models suggest that the effects of adolescent opioids can effect glucose metabolism in future offspring. Indeed, we demonstrated that the adult male offspring of females exposed to morphine for 10 days during adolescence (referred to as MORF1 males) are predisposed to the adverse effects of an obesogenic diet. As adults, MORF1 males fed a high fat moderate sucrose diet (FSD) for just 6 weeks had increased fasting glucose and insulin levels when compared to age-matched offspring of females exposed to saline during adolescence (SALF1 males). Clinically, a similar profile of impaired fasting glucose has been associated with hepatic insulin resistance and an increased risk of non-alcoholic fatty liver disease. Thus, in the current study, we used RNA sequencing to determine whether adult MORF1 males demonstrate significant alterations in the hepatic transcriptome suggestive of alterations in metabolism. Age-matched SALF1 and MORF1 males were fed either FSD or control diet (CD) for 8 weeks. Similar to our previous observations, FSD-maintained MORF1 males gained more weight and displayed both fasting hyperglycemia and hyperinsulinemia when compared to FSD-maintained SALF1 males, with no significant effect on glucagon. No differences in bodyweight or fasting-induce glucose were observed in control diet (CD)-maintained F1 males, although there was a trend for CD MORF1 males to display elevated levels of fasting insulin. Unexpectedly, transcriptional analyses revealed profound differences in the hepatic transcriptome of CD-maintained MORF1 and SALF1 (1686 differentially expressed genes) with no significant differences between FSD-maintained MORF1 and SALF1 males. As changes in the hepatic transcriptome were not revealed under 8 weeks FSD conditions, we extended the feeding paradigm and conducted a glucose tolerance test to determine whether impaired fasting glucose observed in FSD MORF1 males was due to peripheral insulin resistance. Impaired glucose tolerance was observed in both CD and FSD MORF1 males, and to a more limited extent in FSD SALF1 males. These findings implicate intergenerational effects of adolescent morphine exposure on the risk of developing insulin resistance and associated comorbidities, even in the absence of an obesogenic diet.

HPA axis dysfunction during morphine withdrawal in offspring of female rats exposed to opioids preconception.

Opioid use and abuse remain a significant public health problem, particularly in the United States. Indeed, it is estimated that up to 10% of youths (age 12-18) have taken opioids illicitly. A growing body of evidence suggests that this level of widespread opioid exposure can have effects that extend to subsequent generations. Utilizing a well-established rodent model of preconception adolescent opioid exposure in females, we found decreased opioid self-administration coupled with increased cocaine self-administration in adult offspring. This bidirectional effect may be related to negative affect associated with opioid withdrawal, including enhanced stress reactivity. In this study, we tested the hypothesis that the adult offspring of females exposed to morphine during adolescence will demonstrate increased signs of opioid withdrawal when compared to offspring of saline controls. Females were administered increasing doses of morphine (5-25 mg/kg s.c.) or saline (1 ml/kg) from postnatal day 30 (PND30)-PND39. They were then maintained drug free for a minimum of 4 weeks and mated with drug-naïve males on or after PND70. As adults, their male and female offspring (referred to as Mor-F1 or Sal-F1) were administered morphine (10 mg/kg s.c.) twice a day for 5 days. They were then tested for spontaneous withdrawal behaviors for the next 4 days (∼PND70). Levels of corticotropin releasing hormone (Crh) and urocortin 3 (Ucn3) were examined in the amygdala at 48 h and 96 h of withdrawal. Circulating corticosterone was measured at 48 h. Results indicate that Mor-F1 males are heavier than Sal-F1 males with no baseline differences in females. However, Mor-F1 females did not gain weight at the same rate as Sal-F1 females during withdrawal. While there were no differences in somatic withdrawal signs, gene expression data revealed a sex-specific and time-dependent effect on Crh as well as increased Ucn3 and corticosterone in females at 48hrs withdrawal. Overall, these data point to differences in withdrawal and stress reactivity in Mor-F1 animals that may contribute to observed differences in addiction-like behaviors.

Transgenerational effects on anxiety-like behavior following adolescent morphine exposure in female rats.

Global opioid use and misuse remains high, despite efforts to decrease rates of prescribing and diversion. Chronic exposure to opioids, particularly during critical periods of development, can lead to long-lasting effects, including effects that may extend to future generations. Using a rodent model, we have demonstrated significant transgenerational effects of female adolescent morphine exposure, despite the absence of in utero drug exposure. While these effects have been observed in both sexes, effects on anxiety-like behavior were only observed in F1 females. The current study was designed to examine both inter- and transgenerational effects of adolescent morphine exposure on anxiety-like behavior. Female Sprague Dawley rats were administered increasing doses of morphine (5-25 mg/kg s.c.) or saline for 10 days during adolescence (PND30-39). Adult diestrous female offspring (MORF1 or SALF1) and grand offspring (F2) were tested for anxiety-like behavior using the elevated plus maze (EPM). F1 females cross-fostered to donor mothers were also examined. The results show that MORF1 and MORF2 females spend significantly more time on the open arms of the EPM compared to SALF1 controls, an effect that persisted in cross-fostered females. Additional studies demonstrate that this effect is estrous cycle dependent, as decreased anxiety-like behavior was observed in diestrus, while increased anxiety-like behavior was observed in estrus. These behavioral effects were not associated with any differences in circulating corticosterone either at baseline or following EPM testing. Thus, female adolescent morphine exposure alters the regulation of anxiety-like behavior in an estrous-dependent manner and this effect persists in the F2 generation.

A history of opioid exposure in females increases the risk of metabolic disorders in their future male offspring.

Worldwide consumption of opioids remains at historic levels. Preclinical studies report intergenerational effects on the endogenous opioid system of future progeny following preconception morphine exposure. Given the role of endogenous opioids in energy homeostasis, such effects could impact metabolism in the next generation. Thus, we examined diet-induced modifications in F1 male progeny of morphine-exposed female rats (MORF1). When fed a high fat-sugar diet (FSD) for 6 weeks, MORF1 males display features of emerging metabolic syndrome; they consume more food, gain more weight, and develop fasting-induced hyperglycemia and hyperinsulinemia. In the hypothalamus, proteins involved in energy homeostasis are modified and RNA sequencing revealed down-regulation of genes associated with neuronal plasticity, coupled with up-regulation of genes associated with immune, inflammatory, and metabolic processes that are specific to FSD-maintained MORF1 males. Thus, limited preconception morphine exposure in female rats increases the risk of metabolic syndrome/type 2 diabetes in the next generation.

Oxycodone Decreases Dendritic Complexity in Female but not Male Rat Striatal Neurons In Vitro.

The use of oxycodone in the past two decades has dramatically risen, yet the amount of research regarding how it impacts neuronal health is lacking. As prescription use and misuse in women of reproductive age increases there has been a corresponding increase in the number of infants who have been exposed to oxycodone in utero. Given the critical role of the striatum in motor control and reward regulation, the aim of the current study was to examine the effects of oxycodone on developing rat striatal neurons. Sex-specific effects of oxycodone on neuronal cytoarchitecture were examined in cultured rat striatal neurons with a primary focus on dendritic arborization. Neurons were extracted from either male or female embryonic day 18 rat striata and cultured and exposed to varying concentrations of oxycodone over a ten-day period. Dendritic complexity of the neurons was measured using Sholl analysis. Results indicate that oxycodone inhibits dendritic complexity in a dose-dependent manner in female but not male striatal neurons. Additional analysis indicated the number of non-primary dendrites in female striatal neurons significantly decreased with increasing concentrations of oxycodone, while the number of primary dendrites as well as the length of primary and non-primary dendrites was unaffected by oxycodone treatment in both sexes. These in vitro findings demonstrate sex-specific effects of oxycodone on the development of striatal dendritic architecture which may be important for understanding the effects of oxycodone exposure in utero.

Paternal morphine exposure induces bidirectional effects on cocaine versus opioid self-administration.

The United States is in the midst of an opioid epidemic and is thus experiencing unprecedented levels of opioid exposure. A growing body of evidence has demonstrated that this may have consequences on multiple generations. The current set of experiments examined the effect of male adolescent opioid exposure on cocaine and opioid self-administration in the F1 generation. Male Sprague Dawley rats were administered increasing doses of morphine (5-25 mg/kg, s.c.) for 10 days during adolescence (P30-39). Rats were then maintained drug free until adulthood (P70-80) at which point they were mated with drug-naïve females. Male and female F1 offspring were first examined for cocaine self-administration during adulthood. Naïve littermates were tested for morphine self-administration acquisition followed by a within subjects design progressive ratio test for morphine, oxycodone, and cocaine. Results show that male and female F1 rats have delayed acquisition and decreased intake of cocaine. In addition, they have blunted PR levels compared to Sal-F1 control rats. Female Mor-F1 rats also demonstrate increased levels of morphine intake during acquisition and increased PR responding for oxycodone. Surprisingly, even following acquisition of morphine self-administration, Mor-F1 males and females still demonstrate blunted effort for cocaine. There were no differences in sucrose self-administration in naïve littermates. MorF0 seminiferous tubules demonstrated increased levels of acetylated histone H3 and there were increased levels of BDNF mRNA in the mPFC in male and female F1 offspring. Together, these data identify systems that are vulnerable to the impact of opioids in the F0 generation.

Multi-, Inter-, and Transgenerational Effects of Drugs of Abuse on Behavior.

Transgenerational epigenetic inheritance is a burgeoning field that has recently garnered much attention. A growing body of evidence identifies behavioral phenotypes associated with inter-, multi-, and transgenerational studies following a wide variety of parental exposures. This chapter in current topics in behavioral neurogenomics examines the evidence for the presence of behavioral phenotypes and, in particular, the varied and often opposite behavioral responses observed with protocol shifts. Effects following parental exposure to drugs of abuse are used as an example of the wide range of behavioral outcomes and the variability associated with these multiple generation studies. The behavioral phenotypes associated with drug exposure are reviewed in depth.

Challenges to the parental brain: Neuroethological and translational considerations.

Extending from research documenting adaptive parental responses in nonthreatening contexts, the influences of various neuroethological and physiological challenges on effective parenting responses are considered in the current review. In natural habitats, rodent family units are exposed to predators, compromised resources, and other environmental stressors that disrupt HPA axis functions. With the additional physiological demands associated with caring for offspring, alterations in stress-related neuroendocrine responsiveness contribute to adaptive responses in many challenging contexts. Some environmental contexts, however, such as restricted nesting resources, result in disrupted maternal responses that have a negative impact on offspring wellbeing. Additionally, parental dysregulation associated with exposure to environmental chemicals or pharmacological substances, also compromise maternal responses with effects that often extend to future generations. Continued preclinical and clinical research elucidating parental responses to various stressors and physiological disruptors is necessary to provide valuable translational information identifying threats to effective parenting outcomes.

Increased cocaine reward in offspring of females exposed to morphine during adolescence.

RATIONALE: A growing body of evidence demonstrates that environmental exposures can impact the physiology and behavior of subsequent generations. We have previously demonstrated reduced morphine self-administration in the F1 and F2 offspring of female rats exposed to morphine during adolescence. OBJECTIVES: The current study was designed to determine whether attenuated self-administration for a substance not in the opioid class is also observed in the F1 progeny of adolescent morphine exposed females. METHODS: Female adolescent rats were administered morphine at increasing doses for 10 days (P30-39). Females then remained drug free for at least 3 weeks prior to mating with drug-naïve males. As adults, male and female offspring (F1 animals) were tested for cocaine self-administration acquisition, progressive ratio, extinction, and reinstatement. In addition, ß-endorphin peptide levels were measured in the nucleus accumbens (NAc) of behaviorally experienced animals following reinstatement and in behaviorally naïve littermates after acute cocaine (0 or 10 mg/kg, i.p.). Proopiomelanocortin, the polypeptide that is cleaved to produce ß-endorphin, as well as ß-endorphin, was examined in the arcuate nucleus of the hypothalamus and the nucleus accumbens, respectively. Finally, corticosterone was measured following acute cocaine. RESULTS: While no differences were observed during the cocaine acquisition phase (FR-1 and FR-5 schedules), under a PR schedule, Mor-F1 animals (both males and females) had increased motivated responding for cocaine. In addition, Mor-F1 males demonstrated enhanced reinstatement compared to Sal-F1 males. In Mor-F1 males, an acute injection of cocaine (10 mg/kg, i.p.) decreased ß-endorphin levels in the NAc compared to a saline injection while acute cocaine increased ß-endorphin in the NAc in Sal-F1 males compared to saline injection. Following acute cocaine, Mor-F1 males had significantly lower levels of ß-endorphin in the Nac compared to Sal-F1 males. Additionally, ß-endorphin levels in the nucleus accumbens were negatively correlated with reinstatement behavior only in Mor-F1 males. Levels of POMC in the arcuate nucleus were elevated in Mor-F1 males compared to Sal-F1 males, a main effect driven primarily by POMC levels in the acute cocaine condition. These changes were not observed in Mor-F1 females. Finally, plasma corticosterone was increased in Mor-F1 males regardless of acute injection while Mor-F1 females displayed increased corticosterone in response to acute cocaine. CONCLUSIONS: These data indicate that morphine prior to conception increases the rewarding effects of cocaine in male and female offspring. In addition, sex-specific alterations in endogenous opioids and hypothalamic physiology were observed.

Oxycodone self-administration during pregnancy disrupts the maternal-infant dyad and decreases midbrain OPRM1 expression during early postnatal development in rats.

Opioid use and abuse has reached epidemic levels in the United States. As these drugs are frequently used by women of reproductive age, there has been a significant increase in the number of infants born to opioid dependent women. Few preclinical studies have examined voluntary opioid intake during pregnancy, and none have used intravenous self-administration. Thus, the purpose of the current set of studies was to utilize a translational model of oxycodone self-administration in rats to determine the effects of oxycodone intake during pregnancy on early postnatal outcomes. Females were trained to intravenously self-administer oxycodone several weeks prior to mating and then continuously throughout pregnancy followed by withdrawal around the time of parturition. Offspring were monitored for weight gain and separation-induced ultrasonic vocalizations (i.e. number of calls) while dams were examined for motivated maternal responding. Neural expression of the mu opioid receptor gene OPRM1 was examined in offspring on postnatal day 1 (PND1). Results indicate that females self-administer oxycodone during pregnancy at levels similar to those observed in cycling females. Postpartum, oxycodone withdrawn females demonstrate impaired maternal responding. In offspring, while no significant group effects were observed on body weight or call number, age-dependent alterations in weight gain and call number correlated with the dams cumulative oxycodone dose during pregnancy. In addition, offspring demonstrated region specific effects of oxycodone exposure on OPRM1 on PND1. Overall, these findings demonstrate that pregnant females will voluntarily self-administer oxycodone at levels similar to cycling females when using a short access model. Further, maternal oxycodone self-administration alters the maternal-offspring dyad in a manner that is dose-dependent and results in sex- and region-specific effects on OPRM1 expression.

Modeling prenatal opioid exposure in animals: Current findings and future directions.

The past decade has seen a drastic rise in the number of infants exposed to opioids in utero. It is unclear what lasting effect this exposure may have on these children. Animal models of prenatal opioid exposure may provide insight into potential areas of vulnerability. The present review summarizes the findings across animal models of prenatal opioid exposure, including exposure to morphine, methadone, buprenorphine, and oxycodone. Details regarding the drug, doses, and duration of treatment, as well as key findings, are summarized in tables with associated references. Finally, significant gaps in the current preclinical literature and future directions are discussed.

Transgenerational blunting of morphine-induced corticosterone secretion is associated with dysregulated gene expression in male offspring.

A number of parental experiences, even when occurring prior to conception, have been shown to induce transgenerational effects beyond the first generation. In the case of exposure to drugs of abuse, studies in rodents suggest that offspring demonstrate significant differences in how they respond to the drug to which their parent was exposed. We have previously observed significant alterations in morphine analgesia, conditioned place preference and self-administration in the offspring of females exposed to morphine during adolescent development. In addition to effects on pain perception and reward, morphine also modulates the hypothalamic pituitary adrenal (HPA) axis. The purpose of the current study was to determine whether female adolescent morphine exposure results in transgenerational effects on regulation of the HPA axis by morphine in future generations. Adolescent morphine was administered to female Sprague Dawley rats using a 10 day, escalating dose regimen of morphine (5-25 mg/kg; from 30 to 39 days of age). Control animals received saline. Both saline and morphine exposed females (SAL-F0 and MOR-F0, respectively) were mated with drug naïve males beginning at least 3 weeks after the final injection. Plasma corticosterone levels were measured in male and female offspring (F1) during adulthood following 0, 0.1, or 10 mg/kg morphine. In addition, expression of corticotropin releasing hormone (Crh) and mu opioid receptor (Oprm1) in the paraventricular nucleus (PVN) were measured using quantitative PCR. MOR-F1 males, but not females, had blunted morphine-induced corticosterone secretion. This effect was specific to offspring from females exposed to morphine during adolescence as those exposed during adulthood produced offspring in which the effect was absent. In addition, MOR-F1 males had significantly lower levels of PVN Crh following saline. These effects were not driven by PVN oprm1 in the F1 males as there were no differences based on maternal adolescent exposure. To determine the persistence of the blunted morphine-induced corticosterone effect, SAL-F2 and MOR-F2 males were examined. Blunted morphine-induced corticosterone secretion extended into the MOR-F2 generation, as well as effects on Crh. In addition, there was additional dysregulation ofOprm1 expression in the PVN in MOR-F2 compared with SAL-F2 males. These findings suggest that sex-specific alterations in opioid-mediated regulation of the HPA axis are transgenerationally transmitted for at least two generations following female adolescent morphine exposure. These effects may play a role in the previously observed changes in morphine analgesia and reward-related behaviors observed in this phenotype. In addition, alterations in HPA functioning such as these may play a broad role in transgenerational epigenetic transmission.

Transgenerational attenuation of opioid self-administration as a consequence of adolescent morphine exposure.

The United States is in the midst of an opiate epidemic, with abuse of prescription and illegal opioids increasing steadily over the past decade. While it is clear that there is a genetic component to opioid addiction, there is a significant portion of heritability that cannot be explained by genetics alone. The current study was designed to test the hypothesis that maternal exposure to opioids prior to pregnancy alters abuse liability in subsequent generations. Female adolescent Sprague Dawley rats were administered morphine at increasing doses (5-25 mg/kg, s.c.) or saline for 10 days (P30-39). During adulthood, animals were bred with drug-naïve colony males. Male and female adult offspring (F1 animals) were tested for morphine self-administration acquisition, progressive ratio, extinction, and reinstatement at three doses of morphine (0.25, 0.75, 1.25 mg/kg/infusion). Grandoffspring (F2 animals, from the maternal line) were also examined. Additionally, gene expression changes within the nucleus accumbens were examined with RNA deep sequencing (PacBio) and qPCR. There were dose- and sex-dependent effects on all phases of the self-administration paradigm that indicate decreased morphine reinforcement and attenuated relapse-like behavior. Additionally, genes related to synaptic plasticity, as well as myelin basic protein (MBP), were dysregulated. Some, but not all, effects persisted into the subsequent (F2) generation. The results demonstrate that even limited opioid exposure during adolescence can have lasting effects across multiple generations, which has implications for mechanisms of the transmission of drug abuse liability in humans.

Adult Neurogenesis in the Female Mouse Hypothalamus: Estradiol and High-Fat Diet Alter the Generation of Newborn Neurons Expressing Estrogen Receptor α.

Estrogens and leptins act in the hypothalamus to maintain reproduction and energy homeostasis. Neurogenesis in the adult mammalian hypothalamus has been implicated in the regulation of energy homeostasis. Recently, high-fat diet (HFD) and estradiol (E2) have been shown to alter cell proliferation and the number of newborn leptin-responsive neurons in the hypothalamus of adult female mice. The current study tested the hypothesis that new cells expressing estrogen receptor α (ERα) are generated in the arcuate nucleus (ARC) and the ventromedial nucleus of the hypothalamus (VMH) of the adult female mouse, hypothalamic regions that are critical in energy homeostasis. Adult mice were ovariectomized and implanted with capsules containing E2 or oil. Within each hormone group, mice were fed an HFD or standard chow for 6 weeks and treated with BrdU to label new cells. Newborn cells that respond to estrogens were identified in the ARC and VMH, of which a subpopulation was leptin sensitive, indicating that the subpopulation consists of neurons. Moreover, there was an interaction between diet and hormone with an effect on the number of these newborn ERα-expressing neurons that respond to leptin. Regardless of hormone treatment, HFD increased the number of ERα-expressing cells in the ARC and VMH. E2 decreased hypothalamic fibroblast growth factor 10 (Fgf10) gene expression in HFD mice, suggesting a role for Fgf10 in E2 effects on neurogenesis. These findings of newly created estrogen-responsive neurons in the adult brain provide a novel mechanism by which estrogens can act in the hypothalamus to regulate energy homeostasis in females.