Fentanyl-induced acute and conditioned behaviors in two inbred mouse lines: Potential role for Glyoxalase.

An increase in opioid-overdose deaths was evident before the COVID-19 pandemic, and has escalated since its onset. Fentanyl, a highly potent synthetic opioid, is the primary driver of these recent trends. The current study used two inbred mouse strains, C57BL/6 J and A/J, to investigate the genetics of behavioral responses to fentanyl. Mice were tested for conditioned place preference and fentanyl-induced locomotor activity. C57BL/6J mice formed a conditioned place preference to fentanyl injections and fentanyl increased their activity. Neither effect was noted in A/J mice. We conducted RNA-sequencing on the nucleus accumbens of mice used for fentanyl-induced locomotor activity. Surprisingly, we noted few differentially expressed genes using treatment as the main factor. However many genes differed between strains. We validated differences in two genes: suppressor APC domain containing 1 (Sapcd1) and Glyoxalase 1 (Glo1), with quantitative PCR on RNA from the nucleus accumbens and prefrontal cortex (). In both regions A/J mice had significantly higher expression of both genes than did C57BL/6 J. In prefrontal cortex, fentanyl treatment decreased Glo1 mRNA. Glyoxalase 1 catalyzes the detoxification of reactive alpha-oxoaldehydes such as glyoxal and methylglyoxal, is associated with anxiety and activity levels, and its inhibition reduces alcohol intake. We suggest that future studies assess the ability of Glo1 and related metabolites to modify opioid intake.

Perinatal Exposure to Methoxychlor Affects Reproductive Function and Sexual Behavior in Mice.

Numerous chemicals derived from human activity are now disseminated in the environment where their exert estrogenic endocrine disrupting effects, and therefore represent major health concerns. The present study explored whether Methoxychlor (MXC), an insecticide with xenoestrogens activities, given during the perinatal period (from gestational day 11 to postnatal day 8) and at an environmentally dose [20 µg/kg (body weight)/day], would affect reproductive physiology and sexual behavior of the offspring in mice. While MXC exposure did not induce any differences in the weight gain of animals from birth to 4 months of age, a clear difference (although in opposite direction according to the sexes) was observed on the anogenital distance between intact and exposed animals. A similar effect was also observed on preputial separation and vaginal opening, which reflects, respectively, in males and females, puberty occurrence. The advanced puberty observed in females was associated with an enhanced expression of kisspeptin cells in the anteroventral periventricular region of the medial preoptic area. Exposure to MXC did not induce in adult females changes in the estrous cycle or in the weight of the female reproductive tract. By contrast, males showed reduced weight of the epididymis and seminiferous vesicles associated with reduced testosterone levels and seminiferous tubule diameter. We also showed that both males and females showed deficits in mate preference tests. As a whole, our results show that MXC impacts reproductive outcomes.

Sexual Differentiation and Substance Use: A Mini-Review.

The organizational/activational hypothesis suggests that gonadal steroid hormones like testosterone (T) and estradiol (E2) are important at 2 different times during the lifespan when they perform 2 different functions. First steroids "organize" brain structures early in life and during puberty, and in adults these same hormones "activate" sexually dimorphic behaviors. This hypothesis has been tested and proven valid for a large number of behaviors (learning, memory, social, and sexual behaviors). Sex differences in drug addiction are well established both for humans and animal models. Previous research in this field has focused primarily on cocaine self-administration by rats. Traditionally, observed sex differences have been explained by the sex-specific concentrations of gonadal hormones present at the time of the drug-related behavior. Studies with gonadectomized rodents establishes an activational role for E2 that facilitates vulnerability in females, and when E2 is combined with progesterone, addiction is attenuated. Literature on organizational actions of steroids is sparse but predicts that T, after it is aromatized to E2, changes aspects of the neural reward system. Here we summarize these data and propose that sex chromosome complement also plays a role in determining sex-specific drug-taking behavior. Future research is needed to disentangle the effects of hormones and sex chromosome complement, and we propose the four core genotype mouse model as an effective tool for answering these questions.

Sex chromosome complement influences vulnerability to cocaine in mice.

Women acquire cocaine habits faster and are more motivated to obtain drug than men. In general, female rodents acquire intravenous cocaine self-administration (SA) faster and show greater locomotor responses to cocaine than males. Sex differences are attributed to differences in circulating estradiol. We used the four core genotype (FCG) mouse to ask whether sex chromosome complement influences vulnerability to cocaine's reinforcing and/or locomotor-activating effects. The FCG cross produces ovary-bearing mice with XX or XY genotypes (XXF, XYF) and testes-bearing mice with XX or XY genotypes (XXM, XYM). A greater percentage of gonadal females acquired cocaine SA via infusions into jugular catheters as compared with XYM mice, but XXM mice were not significantly different than any other group. Discrimination of the active versus inactive nose poke holes and cocaine intake were in general greater in gonadal females than in gonadal males. Progressive ratio tests for motivation revealed an interaction between sex chromosomes and gonads: XYM mice were more motivated to self-administer cocaine taking more infusions than mice in any other group. Locomotor responses to cocaine exposure revealed effects of sex chromosomes. After acute exposure, activity was greater in XX than in XY mice and the reverse was true for behavioral sensitization. Mice with XY genotypes displayed more activity than XX mice when given cocaine after a 10-day drug-free period. Our data demonstrate that sex chromosome complement alone and/or interacting with gonadal status can modify cocaine's reinforcing and locomotor-activating effects. These data should inform current studies of sex differences in drug use.

Mini-review: Epigenetic mechanisms that promote transgenerational actions of endocrine disrupting chemicals: Applications to behavioral neuroendocrinology.

It is our hope this mini-review will stimulate discussion and new research. Here we briefly examine the literature on transgenerational actions of endocrine disrupting chemicals (EDCs) on brain and behavior and their underlying epigenetic mechanisms including: DNA methylation, histone modifications, and non-coding RNAs. We stress that epigenetic modifications need to be examined in a synergistic manner, as they act together in situ on chromatin to change transcription. Next we highlight recent work from one of our laboratories (VGC). The data provide new evidence that the sperm genome is poised for transcription. In developing sperm, gene enhancers and promoters are accessible for transcription and these activating motifs are also found in preimplantation embryos. Thus, DNA modifications associated with transcription factors during fertilization, in primordial germ cells (PGCs), and/or during germ cell maturation may be passed to offspring. We discuss the implications of this model to EDC exposures and speculate on whether natural variation in hormone levels during fertilization and PGC migration may impart transgenerational effects on brain and behavior. Lastly we discuss how this mechanism could apply to neural sexual differentiation.

Compulsive sucrose- and cocaine-seeking behaviors in male and female Wistar rats.

RATIONALE: Compulsive cocaine use is a key feature of cocaine addiction and understanding the factors that promote the development of such a behavior will provide important insights into the mechanism of cocaine addiction and is essential for the clinical management of the disorder. OBJECTIVES: This study aimed to determine how the preexisting compulsive reward-seeking behavior is related to the development of compulsive cocaine-seeking behavior in male and female rats and the potential impact of the reward value and estrous cycle on such behaviors. METHODS: Adult male and female Wistar rats were first trained to self-administer sucrose pellets under a chained schedule, and then, the intensity-response effects of footshock punishment on sucrose SA reinforced by different values of sucrose were measured. Subsequently, the same rats went on to self-administer intravenous cocaine and the punishment intensity-response effects on cocaine SA reinforced by different doses of cocaine were similarly determined. For the female rats, the measurements were made during different phases of the estrous cycle. RESULTS: The rats showed a wide range of levels of the compulsive behaviors despite the similar training history. Surprisingly, the compulsive sucrose-seeking behavior did not predict the compulsive cocaine-seeking behavior in either sex. Increasing cocaine dose significantly increased the compulsive cocaine-seeking behavior in the female but not male rats. Estrous cycle did not have impact on the compulsive behaviors. CONCLUSION: Preexisting differences in compulsive sucrose-seeking behavior do not predict compulsive cocaine-seeking behavior. Compulsive cocaine-seeking behavior is influenced by cocaine dose but not estrous cycle in the female rats.

Kisspeptin system in ovariectomized mice: Estradiol and progesterone regulation.

The kisspeptin system is clustered in two main groups of cell bodies (the periventricular region, RP3V and the arcuate nucleus, ARC) that send fibers mainly to the GnRH neurons and in a few other locations, including the paraventricular nucleus, PVN. In physiological conditions, gonadal hormones modulate the kisspeptin system with expression changes according to different phases of the estrous cycle: the highest being in estrus phase in RP3V and PVN (positive feedback), and in ARC during the diestrus phase (negative feedback). In this work we wanted to study these hormonal fluctuations during the estrous cycle, investigating the role played by progesterone (P) or estradiol (E2), alone or together, on the kisspeptin system. Gonadectomized CD1 female mice were treated with P, E2 or both (E2 + P), following a timing of administration that emulates the different phases of estrous cycle, for two cycles of 4 days. As expected, the two cell groups were differentially affected by E2; the RP3V group was positively influenced by E2 (alone or with the P), whereas in the ARC the administration of E2 did not affect the system. However P (alone) induced a rise in the kisspeptin immunoreactivity. All the treatments significantly affected the kisspeptin innervation of the PVN, with regional differences, suggesting that these fibers arrive from both RP3V and ARC nuclei.

Different functional domains measured by cocaine self-administration under the progressive-ratio and punishment schedules in male Wistar rats.

BACKGROUND: Current diagnosis of drug addiction like other mental disorders is based on clinical symptoms not on neural pathophysiology and consequently, does not provide useful information on the underlying pathophysiology and may impede the efforts to identify the underlying mechanisms. Identifying the functional deficits that are relevant to addiction and can be traced to the neural systems will greatly facilitate our understanding of the heterogeneity of the condition and improve future diagnosis and treatment. Cocaine addiction is characterized by the continued use despite the dire consequences, and the deficit in inhibitory control may play a key role in this process. This study aimed to develop a paradigm to measure the punishment-induced inhibitory regulation of reward-seeking behavior. METHODS: Rats were first trained to self-administer sucrose pellets under a chained schedule and then the breaking points (BPs) under the progressive-ratio schedule, and the intensity-response effects of footshock punishment on sucrose SA were measured. Subsequently, the rats went on to self-administer intravenous cocaine, and the BPs and the punishment intensity-response effects were similarly determined. RESULTS: The areas under the punishment intensity-response curves (AUCs) were calculated and used as an indicator of the sensitivity of the inhibitory system. The BPs for cocaine were not correlated with the AUCs. Furthermore, the change in the BPs for cocaine induced by changing cocaine dose did not predict the change in the AUCs. CONCLUSION: The intensity-response effects of punishment can be used to measure the function or sensitivity of the inhibitory system independent of the motivational state.

Sex Differences in the Motivational Contrast between Sucrose and Cocaine in Rats.

There are sex differences in the vulnerability to cocaine abuse and addiction. Understanding the differences is critical for developing the sex-tailored prevention and treatment strategies. Cocaine addiction is characterized by the pathological motivation for cocaine accompanied by the diminished motivation for natural rewards. Thus, the motivational impact of cocaine relative to natural rewards likely determines the attractiveness of cocaine and likely plays a role in the vulnerability to cocaine abuse and addiction. This study aimed to determine whether the relative magnitudes or contrast of the motivational impact between cocaine and sucrose is different between sexes. To this end, cocaine-naïve out bred Wistar rats were trained to self-administer sucrose pellets and the motivation for different amounts of sucrose was then determined as the breakpoints under the progressive-ratio schedule of reinforcement. Following the sucrose tests, the same rats were trained to self-administer cocaine and the motivation for different doses of cocaine was similarly measured. For the female rats, the motivation was also measured during the diestrus and proestrus/estrus, respectively, to determine the impact of the estrous cycle on the motivational effects of cocaine and sucrose. The differences between the breakpoints of cocaine and sucrose were significantly larger in the males. The enhanced motivational contrast may contribute to the increased vulnerability to recreational cocaine abuse and addiction in the males.

Revisiting the neural role of estrogen receptor beta in male sexual behavior by conditional mutagenesis.

Estradiol derived from neural aromatization of gonadal testosterone plays a key role in the perinatal organization of the neural circuitry underlying male sexual behavior. The aim of this study was to investigate the contribution of neural estrogen receptor (ER) ß in estradiol-induced effects without interfering with its peripheral functions. For this purpose, male mice lacking ERß in the nervous system were generated. Analyses of males in two consecutive tests with a time interval of two weeks showed an effect of experience, but not of genotype, on the latencies to the first mount, intromission, pelvic thrusting and ejaculation. Similarly, there was an effect of experience, but not of genotype, on the number of thrusts and mating length. Neural ERß deletion had no effect on the ability of males to adopt a lordosis posture in response to male mounts, after castration and priming with estradiol and progesterone. Indeed, only low percentages of both genotypes exhibited a low lordosis quotient. It also did not affect their olfactory preference. Quantification of tyrosine hydroxylase- and kisspeptin-immunoreactive neurons in the preoptic area showed unaffected sexual dimorphism of both populations in mutants. By contrast, the number of androgen receptor- and ERα-immunoreactive cells was significantly increased in the bed nucleus of stria terminalis of mutant males. These data show that neural ERß does not play a crucial role in the organization and activation of the neural circuitry underlying male sexual behavior. These discrepancies with the phenotype of global ERß knockout models are discussed.

Developmental Exposure to Ethinylestradiol Affects Reproductive Physiology, the GnRH Neuroendocrine Network and Behaviors in Female Mouse.

During development, environmental estrogens are able to induce an estrogen mimetic action that may interfere with endocrine and neuroendocrine systems. The present study investigated the effects on the reproductive function in female mice following developmental exposure to pharmaceutical ethinylestradiol (EE2), the most widespread and potent synthetic steroid present in aquatic environments. EE2 was administrated in drinking water at environmentally relevant (ENVIR) or pharmacological (PHARMACO) doses [0.1 and 1 µg/kg (body weight)/day respectively], from embryonic day 10 until postnatal day 40. Our results show that both groups of EE2-exposed females had advanced vaginal opening and shorter estrus cycles, but a normal fertility rate compared to CONTROL females. The hypothalamic population of GnRH neurons was affected by EE2 exposure with a significant increase in the number of perikarya in the preoptic area of the PHARMACO group and a modification in their distribution in the ENVIR group, both associated with a marked decrease in GnRH fibers immunoreactivity in the median eminence. In EE2-exposed females, behavioral tests highlighted a disturbed maternal behavior, a higher lordosis response, a lack of discrimination between gonad-intact and castrated males in sexually experienced females, and an increased anxiety-related behavior. Altogether, these results put emphasis on the high sensitivity of sexually dimorphic behaviors and neuroendocrine circuits to disruptive effects of EDCs.

Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta.

Ovarian oestradiol is essential for pubertal maturation and adult physiology of the female reproductive axis. It acts at central and peripheral sites through two main oestrogen receptors (ER) α and ß. Here we investigate the role of ERß on central effects of oestradiol, by generating a mouse line specifically lacking the ERß gene in neuronal and glial cells. Central ERß deletion delays the age at vaginal opening and first oestrous and reduces uterine weight without affecting body growth. Analysis of factors necessary for pubertal progression shows reduced levels of Kiss1 transcripts at postnatal (P) day 25 in the preoptic area, but not in the mediobasal hypothalamus (MBH) of mutant females. In agreement with these data, the number of kisspeptin-immunoreactive neurons was decreased by 57-72% in the three subdivisions of the rostral periventricular area of the third ventricle (RP3V), whereas the density of kisspeptin-immunoreactive fibres was unchanged in the arcuate nucleus of mutant mice. These alterations do not involve changes in ERα mRNAs in the preoptic area and protein levels in the RP3V. The number and distribution of GnRH-immunoreactive cells were unaffected, but gonadotropin-releasing hormone (GnRH) transcript levels were higher in the P25 preoptic area of mutants. At adulthood, mutant females have normal oestrous cyclicity, kisspeptin system and exhibit unaltered sexual behaviour. They display, however, reduced ovary weight and increased anxiety-related behaviour during the follicular phase. This argues for the specific involvement of central ERß in the regulation of pubertal onset in female reproduction, possibly through prepubertal induction of kisspeptin expression in the RP3V.

Perinatal exposure to methoxychlor enhances adult cognitive responses and hippocampal neurogenesis in mice.

During perinatal life, sex steroids, such as estradiol, have marked effects on the development and function of the nervous system. Environmental estrogens or xenoestrogens are man-made chemicals, which animal and human population encounter in the environment and which are able to disrupt the functioning of the endocrine system. Scientific interest in the effects of exposure to xenoestrogens has focused more on fertility and reproductive behaviors, while the effects on cognitive behaviors have received less attention. Therefore, the present study explored whether the organochlorine insecticide Methoxychlor (MXC), with known xenoestrogens properties, administered during the perinatal period (from gestational day 11 to postnatal day 8) to pregnant-lactating females, at an environmentally relevant dose (20 µg/kg (body weight)/day), would also affect learning and memory functions depending on the hippocampus of male and female offspring mice in adulthood. When tested in adulthood, MXC perinatal exposure led to an increase in anxiety-like behavior and in short-term spatial working memory in both sexes. Emotional learning was also assessed using a contextual fear paradigm and MXC treated male and female mice showed an enhanced freezing behavior compared to controls. These results were correlated with an increased survival of adult generated cells in the adult hippocampus. In conclusion, our results show that perinatal exposure to an environmentally relevant dose of MXC has an organizational effect on hippocampus-dependent memory and emotional behaviors.

Estrous cycle and sex affect cocaine-induced behavioural changes in CD1 mice.

RATIONALE: Several findings on sex differences in cocaine response suggest a role for hormonal milieu in modulating the subjective effects of cocaine. Nitric oxide (NO) has been involved in the neurochemical, hormonal, and behavioral changes related to stress and anxiety. Within the brain, the anteroventral subdivision of the medial amygdala (MeAV) is an important area involved in processing emotional responses such as anxiety and a high density of NO-producing neurons is observed in this area. OBJECTIVES: In this study, we hypothesize the possibility of sex/hormonal differences in response to cocaine and that these differences may reflect a change in the MeAV nitrergic system. We have examined cocaine's acute effects on nicotinamide adenine dinucleotide phosphate diaphorase (nadph-d) expression, as well as its effect on motor activity and anxiety in male and estrus and diestrus females. RESULTS: Our results show that acute cocaine administration produces an increase in both anxiety behaviors and nadph-d expression in the MeAV. Male and diestrus female mice were more susceptible to these effects of cocaine than estrus female mice in which no differences were detected. In addition, we examined individual differences in male and female mice responding to intravenous cocaine reinforcement in a self-administration paradigm. Female mice acquired cocaine self-administration at a faster rate than males and showed a higher motivation to self-administer cocaine under a progressive ratio schedule of reinforcement. CONCLUSIONS: Our data suggest a complex interaction between hormonal milieu and the behavioral and reinforcing effects of cocaine.

Neuroendocrine and behavioral effects of maternal exposure to oral bisphenol A in female mice.

Bisphenol A (BPA) is a widespread estrogenic compound. We investigated the effects of maternal exposure to BPA at reference doses on sexual behavior and neuroendocrine functions of female offspring in C57BL/6J mice. The dams were orally exposed to vehicle alone or vehicle-containing BPA at doses equivalent to the no observed adverse effect level (5 mg/kg body weight per day) and tolerable daily intake (TDI, 0.05 mg/kg body weight per day) level from gestational day 15 until weaning. Developmental exposure to BPA increased the lordosis quotient in naive females exposed to BPA at the TDI dose only. BPA exposure had no effect on olfactory preference, ability to express masculine behaviors or number of calbindin-positive cells, a sexually dimorphic population of the preoptic area. BPA at both doses selectively increased kisspeptin cell number in the preoptic periventricular nucleus of the rostral periventricular area of the third ventricle in adult females. It did not affect the number of GNRH-positive cells or percentage of kisspeptin appositions on GNRH neurons in the preoptic area. These changes were associated with higher levels of estradiol (E2) at the TDI dose while levels of LH, estrus cyclicity, ovarian and uterine weights, and fertility remained unaffected. Delay in the time of vaginal opening was observed during the postnatal period at TDI dose, without any alteration in body growth. This shows that developmental exposure to BPA at reference doses did not masculinize and defeminize the neural circuitry underlying sexual behavior in female mice. The TDI dose specifically exacerbated responses normally induced by ovarian E2, through estrogen receptor α, during the postnatal/prepubertal period.

Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol a in male mice.

There are human reproduction concerns associated with extensive use of bisphenol A (BPA)-containing plastic and, in particular, the leaching of BPA into food and beverages. In this context, it remains unclear whether and how exposure to BPA interferes with the developmental organization and adult activation of male sexual behavior by testosterone. We evaluated the developmental and adult exposure to oral BPA at doses equivalent to the no-observed-adverse-effect-level (5 mg/kg body weight per day) and tolerable daily intake (TDI) (50 µg/kg body weight per day) on mouse sexual behavior and the potential mechanisms underlying BPA effects. Adult exposure to BPA reduced sexual motivation and performance at TDI dose only. Exposed males took longer to initiate mating and reach ejaculation despite normal olfactory chemoinvestigation. This deficiency was not restored by sexual experience and was associated with unchanged circulating levels of testosterone. By contrast, developmental exposure to BPA at TDI or no-observed-adverse-effect-level dose did not reduce sexual behavior or alter the neuroanatomical organization of the preoptic area. Disrupting the neural androgen receptor resulted in behavioral and neuroanatomical effects similar to those induced by adult exposure to TDI dose. Moreover, adult exposure of mutant males to BPA at TDI dose did not trigger additional alteration of sexual behavior, suggesting that BPA and neural androgen receptor mutation share a common mechanism of action. This shows, for the first time, that the neural circuitry underlying male sexual behavior is vulnerable to chronic adult exposure to low dose of BPA and suggests that BPA could act in vivo as an antiandrogenic compound.

Kisspeptin: a new neuronal target of primer pheromones in the control of reproductive function in mammals.

Pheromones are known to trigger either short-term behavioral responses, usually referred to as "releaser effects", or more long-term physiological changes, known as "primer effects", which especially affect reproductive function at the level of the gonadotrope axis. The precise mechanisms through which pheromones interact with the gonadotrope axis in the hypothalamus is not fully known. We propose that the neuropeptide Kisspeptin, could be a specific target of primer pheromones, allowing these pheromones to modulate the gonadotrope axis and GnRH activity. This emerging hypothesis is discussed in the context of puberty acceleration in female mice and the male effect in female ungulates (sheep or goat). These examples have been chosen to illustrate the diversity of the reproductive contexts in mammals and potential mechanisms affected by primer effects at the level of the gonadotrope axis.

A single episode of maternal deprivation impairs the motivation for cocaine in adolescent mice.

RATIONALE: Early-life adverse events, like maternal deprivation (MD), have been associated with the later development of mood and anxiety disorders. Scarce data are available describing behavioural and endocrine alterations in maternally deprived (DEP) animals during the periadolescent period. We hypothesize that a single episode of MD early in life would alter reward function and lead to a long-lasting behavioural and neuroendocrine changes during adolescence. OBJECTIVES: Our aim was to evaluate the effects of a single episode of MD in CD1 adolescent mice (postnatal day 35) on a range of tests for anxiety- and depression-related behaviours (open field, elevated plus maze and tail suspension test). We further assess whether these effects could affect cocaine self-administration behaviour. In order to correlate behavioural and neuroendocrine responses to stress, brain-derived neurotrophic factor (BDNF) levels were assessed in brain structures related to emotional and cognitive processes. RESULTS: During the cocaine self-administration, the time required for achieving the acquisition criteria was significantly increased and the breaking point values in progressive schedule were significantly reduced in DEP adolescent mice, suggesting impairment in rewarding functions. The behavioural tests also confirm an increase in anxiety- and depression-related behaviours in DEP adolescent mice. The results on BDNF level indicated a decrease in response to MD in amygdala and hippocampus, confirming the behavioural data. CONCLUSIONS: Our findings demonstrated for the first time that a single episode of early MD can impair the motivation for cocaine consumption in adolescent mice and can be associated with anxiety- and depressive-like behaviour.

Synergic effects of estradiol and progesterone on regulation of the hypothalamic neuronal nitric oxide synthase expression in ovariectomized mice.

Nitric oxide (NO) is a gaseous neurotransmitter that plays an important role in the regulation of sexual behavior in rodents. NO is produced, within the central nervous system, by the enzyme neural NO synthase (nNOS) whose expression is influenced by gonadal hormones. In previous studies, we demonstrated that part of the nitrergic hypothalamic and limbic system is influenced, in physiological conditions, by the hormonal fluctuations during the estrous cycle, but we were unable to distinguish among the role played by progesterone (P) or estradiol (E(2)) in inducing these changes. In the present study, we investigated the effects of E(2) and P (alone or together) on the nitrergic system of gonadectomized female mice, following a timing of administration that emulates the different phases of estrous cycle. In parallel, we tested the influence of the two hormones on sexual behavior, confirming that P works in synergistic fashion with E(2) to facilitate female receptivity. The quantitative analysis of nNOS-ir system demonstrated a statistically significant variation in the number of positive cells only in those part of the limbic-hypothalamic nitrergic system that are affected in cycling females, i.e. the bed nucleus of the stria terminalis, the arcuate nucleus and the medial preoptic area, with the highest number of positive neurons observed in E(2)+P group. The variable effects of E(2) and P may depend on the different distribution of their receptors within the analyzed nuclei, but the relationships among variations of estrogen and progesterone levels and in vivo modulation of nNOS expression remain unknown and needed further investigations.