Bisphenol-A induces expression of HOXC6, an estrogen-regulated homeobox-containing gene associated with breast cancer.

HOXC6 is a homeobox-containing gene associated with mammary gland development and is overexpressed in variety of cancers including breast and prostate cancers. Here, we have examined the expression of HOXC6 in breast cancer tissue, investigated its transcriptional regulation via estradiol (E2) and bisphenol-A (BPA, an estrogenic endocrine disruptor) in vitro and in vivo. We observed that HOXC6 is differentially over-expressed in breast cancer tissue. E2 induces HOXC6 expression in cultured breast cancer cells and in mammary glands of Sprague Dawley rats. HOXC6 expression is also induced upon exposure to BPA both in vitro and in vivo. Estrogen-receptor-alpha (ERα) and ER-coregulators such as MLL-histone methylases are bound to the HOXC6 promoter upon exposure to E2 or BPA and that resulted in increased histone H3K4-trimethylation, histone acetylation, and recruitment of RNA polymerase II at the HOXC6 promoter. HOXC6 overexpression induces expression of tumor growth factors and facilitates growth 3D-colony formation, indicating its potential roles in tumor growth. Our studies demonstrate that HOXC6, which is a critical player in mammary gland development, is upregulated in multiple cases of breast cancer, and is transcriptionally regulated by E2 and BPA, in vitro and in vivo.

Dynamic regulation of BDNF gene expression by estradiol and lncRNA HOTAIR.

Brain derived neurotrophic factor (BDNF) is a major neurotransmitter that controls growth and maintenance of neurons and its misregulation is linked to neurodegeneration and human diseases. Estradiol (E2) is well-known to regulate the process of differentiation and plasticity of hippocampal neurons. Here we examined the mechanisms of BDNF gene regulation under basal conditions and under stimuli such as E2. Our results demonstrated that BDNF expression is induced by E2 in vitro in HT22 cells (hippocampal neuronal cells) and in vivo (in ovariectomized mouse brain under E2-treatment). Using chromatin immunoprecipitation assay, we demonstrated that estrogen receptors (ERα, ERß) were enriched at the BDNF promoter in presence of E2. Additionally, ER-coregulators (e.g., CBP/p300, MLL3), histone acetylation, H3K4-trimethylation, and RNA polymerase II levels were also elevated at the BDNF promoter in an E2-dependent manner. Additionally, under the basal conditions (in the absence of E2), the long noncoding RNA HOTAIR and its interacting partners PRC2 and LSD1 complexes binds to the promoter of BDNF and represses its expression. HOTAIR knockdown -relieves the repression resulting in elevation of BDNF expression. Further, levels of HOTAIR-interacting partners, EZH2 and LSD1 were reduced at the BDNF promoter upon HOTAIR-knockdown revealing that HOTAIR plays a regulatory role in BDNF gene expression by modulating promoter histone modifications. Additionally, we showed that E2 induced-BDNF expression is mediated by the displacement of silencing factors, EZH2 and LSD1 at BDNF promoter and subsequent recruitment of active transcription machinery. These results reveal the mechanisms of BDNF gene regulation under the basal condition and in presence of a positive regulator such as E2 in neuronal cells.

Interactions between estradiol and ERK, but not mTOR, signaling is necessary for enhanced cocaine-induced conditioned place preference in female rats.

Women rapidly progress from recreational cocaine use to dependence, consume greater quantities of cocaine, experience more positive subjective effects of cocaine and have higher incidences of relapse during abstinence. These effects have been replicated in animal models of cocaine addiction and indicate an enhanced sensitivity and therefore, vulnerability of females to cocaine addiction. Furthermore, it has been demonstrated that estradiol (E2) is a key mediator of the aforementioned effects of cocaine in women and female animals. However, studies identifying the influence of E2 on cocaine-associated reward and its underlying neurobiological mechanisms are lacking. Here, we further explored the influence of E2 on cocaine conditioned place preference in female rats. We show that E2 mediates cocaine-conditioned reward by potentiating cocaine-context associations. In addition, the E2-mediated increases in cocaine-induced CPP are associated with increased activation of ERK1/2 and mTOR proteins in the nucleus accumbens, dorsal striatum, and ventral tegmental area. To assess the involvement of ERK1/2 and mTOR in E2-mediated enhanced cocaine-CPP, we inhibited ERK1/2 and/or mTOR activity during cocaine-conditioning and before CPP-test. Inhibition of ERK1/2 during conditioning blocked cocaine-CPP in females, inhibition mTOR was without effect, and inhibiting ERK1/2 and mTOR before CPP-test blocked cocaine-CPP. In conclusion, we have established that E2 enhances cocaine-conditioned reward by potentiating cocaine-context associations formed during conditioning. Additionally, activation of ERK1/2 during cocaine-conditioning is necessary for the potentiation of cocaine-conditioned reward by E2. SIGNIFICANCE STATEMENT: Studies characterizing the molecular substrates underlying the effects of E2 during the formation of cocaine-context associations are virtually unknown. In this study, we established the influence of E2 during the formation of cocaine-CPP and characterized the role of ERK1/2 and mTOR activity on this effect within significant nodes of the reward pathway. The elucidation of the role of E2 in cocaine-induced intracellular signaling fills a significant gap in our knowledge regarding the mechanisms by which E2 affects intracellular signaling pathways to indicate the motivational salience of a stimulus. These data are crucial to our understanding of how fluctuating hormone levels can render females increasing sensitive to the rewarding effects of cocaine.

Nuclear factor kappa B signaling regulates neuronal morphology and cocaine reward.

Although chronic cocaine-induced changes in dendritic spines on nucleus accumbens (NAc) neurons have been correlated with behavioral sensitization, the molecular pathways governing these structural changes, and their resulting behavioral effects, are poorly understood. The transcription factor, nuclear factor kappa B (NFkappaB), is rapidly activated by diverse stimuli and regulates expression of many genes known to maintain cell structure. Therefore, we evaluated the role of NFkappaB in regulating cocaine-induced dendritic spine changes on medium spiny neurons of the NAc and the rewarding effects of cocaine. We show that chronic cocaine induces NFkappaB-dependent transcription in the NAc of NFkappaB-Lac transgenic mice. This induction of NFkappaB activity is accompanied by increased expression of several NFkappaB genes, the promoters of which show chromatin modifications after chronic cocaine exposure consistent with their transcriptional activation. To study the functional significance of this induction, we used viral-mediated gene transfer to express either a constitutively active or dominant-negative mutant of Inhibitor of kappa B kinase (IKKca or IKKdn), which normally activates NFkappaB signaling, in the NAc. We found that activation of NFkappaB by IKKca increases the number of dendritic spines on NAc neurons, whereas inhibition of NFkappaB by IKKdn decreases basal dendritic spine number and blocks the increase in dendritic spines after chronic cocaine. Moreover, inhibition of NFkappaB blocks the rewarding effects of cocaine and the ability of previous cocaine exposure to increase an animal's preference for cocaine. Together, these studies establish a direct role for NFkappaB pathways in the NAc to regulate structural and behavioral plasticity to cocaine.

Sex Differences and the Role of Estradiol in Mesolimbic Reward Circuits and Vulnerability to Cocaine and Opiate Addiction.

Although both men and women become addicted to drugs of abuse, women transition to addiction faster, experience greater difficulties remaining abstinent, and relapse more often than men. In both humans and rodents, hormonal cycles are associated with females' faster progression to addiction. Higher concentrations and fluctuating levels of ovarian hormones in females modulate the mesolimbic reward system and influence reward-directed behavior. For example, in female rodents, estradiol (E2) influences dopamine activity within the mesolimbic reward system such that drug-directed behaviors that are normally rewarding and reinforcing become enhanced when circulating levels of E2 are high. Therefore, neuroendocrine interactions, in part, explain sex differences in behaviors motivated by drug reward. Here, we review sex differences in the physiology and function of the mesolimbic reward system in order to explore the notion that sex differences in response to drugs of abuse, specifically cocaine and opiates, are the result of molecular neuroadaptations that differentially develop depending upon the hormonal state of the animal. We also reconsider the notion that ovarian hormones, specifically estrogen/estradiol, sensitize target neurons thereby increasing responsivity when under the influence of either cocaine or opiates or in response to exposure to drug-associated cues. These adaptations may ultimately serve to guide the motivational behaviors that underlie the factors that cause women to be more vulnerable to cocaine and opiate addiction than men.

Overlap in the neural circuitry and molecular mechanisms underlying ketamine abuse and its use as an antidepressant.

Ketamine, a dissociative anesthetic and psychedelic compound, has revolutionized the field of psychopharmacology by showing robust, and rapid-acting antidepressant activity in patients suffering from major depressive disorder (MDD), suicidal tendencies, and treatment-resistant depression (TRD). Ketamine's efficacy, however, is transient, and patients must return to the clinic for repeated treatment as they experience relapse. This is cause for concern because ketamine is known for its abuse liability, and repeated exposure to drugs of abuse often leads to drug abuse/dependence. Though the mechanism(s) underlying its antidepressant activity is an area of current intense research, both clinical and preclinical evidence shows that ketamine's effects are mediated, at least in part, by molecular adaptations resulting in long-lasting synaptic changes in mesolimbic brain regions known to regulate natural and drug reward. This review outlines our limited knowledge of ketamine's neurobiological and biochemical underpinnings mediating its antidepressant effects and correlates them to its abuse potential. Depression and addiction share overlapping neural circuitry and molecular mechanisms, and though speculative, repeated use of ketamine for the treatment of depression could lead to the development of substance use disorder/addiction, and thus should be tempered with caution. There is much that remains to be known about the long-term effects of ketamine, and our lack of understanding of neurobiological mechanisms underlying its antidepressant effects is a clear limiting factor that needs to be addressed systematically before using repeated ketamine in the treatment of depressed patients.

HOXA5 Expression Is Elevated in Breast Cancer and Is Transcriptionally Regulated by Estradiol.

HOXA5 is a homeobox-containing gene associated with the development of the lung, gastrointestinal tract, and vertebrae. Here, we investigate potential roles and the gene regulatory mechanism in HOXA5 in breast cancer cells. Our studies demonstrate that HOXA5 expression is elevated in breast cancer tissues and in estrogen receptor (ER)-positive breast cancer cells. HOXA5 expression is critical for breast cancer cell viability. Biochemical studies show that estradiol (E2) regulates HOXA5 gene expression in cultured breast cancer cells in vitro. HOXA5 expression is also upregulated in vivo in the mammary tissues of ovariectomized female rats. E2-induced HOXA5 expression is coordinated by ERs. Knockdown of either ERα or ERß downregulated E2-induced HOXA5 expression. Additionally, ER co-regulators, including CBP/p300 (histone acetylases) and MLL-histone methylases (MLL2, MLL3), histone acetylation-, and H3K4 trimethylation levels are enriched at the HOXA5 promoter in present E2. In summary, our studies demonstrate that HOXA5 is overexpressed in breast cancer and is transcriptionally regulated via estradiol in breast cancer cells.

The influence of DeltaFosB in the nucleus accumbens on natural reward-related behavior.

The transcription factor deltaFosB (DeltaFosB), induced in nucleus accumbens (NAc) by chronic exposure to drugs of abuse, has been shown to mediate sensitized responses to these drugs. However, less is known about a role for DeltaFosB in regulating responses to natural rewards. Here, we demonstrate that two powerful natural reward behaviors, sucrose drinking and sexual behavior, increase levels of DeltaFosB in the NAc. We then use viral-mediated gene transfer to study how such DeltaFosB induction influences behavioral responses to these natural rewards. We demonstrate that overexpression of DeltaFosB in the NAc increases sucrose intake and promotes aspects of sexual behavior. In addition, we show that animals with previous sexual experience, which exhibit increased DeltaFosB levels, also show an increase in sucrose consumption. This work suggests that DeltaFosB is not only induced in the NAc by drugs of abuse, but also by natural rewarding stimuli. Additionally, our findings show that chronic exposure to stimuli that induce DeltaFosB in the NAc can increase consumption of other natural rewards.

Sex differences in the expression of morphine withdrawal symptoms and associated activity in the tail of the ventral tegmental area.

Recent studies, in male rodents, have begun to elucidate a role for the GABAergic neurons in the tail of the ventral tegmental area (tVTA) in morphine withdrawal. To date, the mechanisms underlying morphine withdrawal have been studied almost exclusively in male animals. As a result, there is a considerable gap in our current understanding of the processes underlying sex differences in morphine withdrawal behaviors and its effects on cellular activity in the tVTA in females. The purpose of the present study was to investigate the influence of sex on the expression and duration of spontaneous somatic morphine withdrawal syndrome, and to characterize the relationship between spontaneous somatic withdrawal symptoms and cellular activation (measured as phosphorylated CREB; pCREB), in the GABAergic tVTA in male and female rats. Morphine-dependent adult male and female Long Evans rats underwent 72 h of spontaneous withdrawal, and somatic withdrawal symptoms were assessed every 12 h. Male morphine-dependent rats expressed more severe symptoms during the early phases of withdrawal compared to females. Although, females demonstrated lower overall symptom severity, their symptoms persisted for a longer period of time, thus demonstrating higher withdrawal-symptom severity than males during late withdrawal. pCREB activity in the tVTA was elevated in morphine-withdrawn rats and was positively correlated with the severity of withdrawal symptoms. These results demonstrate sex differences in the timing of the expression of somatic withdrawal. Our data add to the growing body of evidence demonstrating a role for the tVTA in morphine withdrawal and begin to establish a sex-dependent behavioral and molecular profile within this brain region.

DeltaFosB induction in orbitofrontal cortex mediates tolerance to cocaine-induced cognitive dysfunction.

Current cocaine users show little evidence of cognitive impairment and may perform better when using cocaine, yet withdrawal from prolonged cocaine use unmasks dramatic cognitive deficits. It has been suggested that such impairments arise in part through drug-induced dysfunction within the orbitofrontal cortex (OFC), yet the neurobiological mechanisms remain unknown. We observed that chronic cocaine self-administration increased expression of the transcription factor deltaFosB within both medial and orbitofrontal regions of the rat prefrontal cortex. However, the increase in OFC deltaFosB levels was more pronounced after self-administered rather than experimenter-administered cocaine, a pattern that was not observed in other regions. We then used rodent tests of attention and decision making to determine whether deltaFosB within the OFC contributes to drug-induced alterations in cognition. Chronic cocaine treatment produced tolerance to the cognitive impairments caused by acute cocaine. Overexpression of a dominant-negative antagonist of deltaFosB, deltaJunD, in the OFC prevented this behavioral adaptation, whereas locally overexpressing deltaFosB mimicked the effects of chronic cocaine. Gene microarray analyses identified potential molecular mechanisms underlying this behavioral change, including an increase in transcription of metabotropic glutamate receptor subunit 5 and GABA(A) receptors as well as substance P. Identification of deltaFosB in the OFC as a mediator of tolerance to the effects of cocaine on cognition provides fundamentally new insight into the transcriptional modifications associated with addiction.

Postnatal loss of methyl-CpG binding protein 2 in the forebrain is sufficient to mediate behavioral aspects of Rett syndrome in mice.

BACKGROUND: Mutations in the methyl-CpG binding protein 2 (MeCP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder that is accompanied by a broad array of behavioral phenotypes, mainly affecting females. Methyl-CpG binding protein 2 is a transcriptional repressor that is widely expressed in all tissues. METHODS: To investigate whether the postnatal loss of MeCP2 in the forebrain is sufficient to produce the behavioral phenotypes observed in RTT, we have generated conditional MeCP2 knockout mice. RESULTS: These mice display behavioral abnormalities similar to RTT phenotypes, including hindlimb clasping, impaired motor coordination, increased anxiety, and abnormal social behavior with other mice. These mice, however, have normal locomotor activity and unimpaired context-dependent fear conditioning, suggesting that the behavioral deficits observed are the result of loss of MeCP2 function in postnatal forebrain and not the result of generalized global deficits. CONCLUSIONS: These data highlight the important role of MeCP2 in the forebrain and suggest that even partial loss of MeCP2 expression in these brain regions is sufficient to recapitulate features of RTT.

Exposure to morphine-associated cues increases mu opioid receptor mRNA expression in the nucleus accumbens of Wistar Kyoto rats.

The Wistar-Kyoto (WKY) rat has been proposed as a model of anxiety vulnerability as it exhibits pronounced behavioral inhibition, passive avoidance, exaggerated startle response, enhanced HPA-axis activation, and active avoidance that is resistant to extinction. Accumulating evidence suggests that WKY rats respond differently to rewarding stimuli when compared to outbred strains of rat. Conditioned responding to drug-associated cues is linked with alterations in the activation of mu opioid receptors (MOR) and kappa opioid receptors (KOR) in the nucleus accumbens (NAc). Furthermore, alterations in KOR expression/activation in the NAc of WKY rats are implicated in the regulation of some of the components that make up the unique behavioral phenotype of this strain. The purpose of this study was to extend upon previous work from our laboratory by investigating conditioned morphine reward in adult male WKY and SD rats, and to examine levels of KOR mRNA and MOR mRNA in the NAc at baseline and after acquisition of morphine CPP. Our results demonstrate that SD rats displayed morphine-induced CPP to each of the six doses of morphine tested (0.5, 1.25, 2.5, 5, 7.5, or 10mg/kg). Interestingly, WKY rats demonstrated CPP for only the 1.25, 2.5, and 5mg/kg doses, yet no preference at the lowest (0.5mg/kg) or highest (7.5 and 10mg/kg) doses. qPCR analysis of MOR and KOR in the NAc revealed no strain differences in basal levels of MOR, but higher levels of KOR in WKY rats compared to those of SD rats. Interestingly, after completion of the CPP task, WKY rats had overall higher levels of NAc MOR mRNA compared to SD rats; the initial basal differences in NAc KOR levels persisted without change due to CPP in either strain. These results demonstrate that the WKY rat exhibits a unique pattern of behavioral responding to morphine and implicates differences in NAc KOR signaling as a potential source of aversion to higher doses of morphine. Additionally, the CPP-induced upregulation of NAc MOR mRNA in WKY rats warrants further investigation in terms of its potential role as a factor constituting a unique vulnerability to subsequent drug exposure.

Local field potentials in the ventral tegmental area during cocaine-induced locomotor activation: Measurements in freely moving rats.

The ventral tegmental area (VTA) has been established as a critical nucleus for processing behavioral changes that occur during psychostimulant use. Although it is known that cocaine induced locomotor activity is initiated in the VTA, not much is known about the electrical activity in real time. The use of our custom-designed wireless module for recording local field potential (LFP) activity provides an opportunity to confirm and identify changes in neuronal activity within the VTA of freely moving rats. The purpose of this study was to investigate the changes in VTA LFP activity in real time that underlie cocaine induced changes in locomotor behavior. Recording electrodes were implanted in the VTA of rats. Locomotor behavior and LFP activity were simultaneously recorded at baseline, and after saline and cocaine injections. Results indicate that cocaine treatment caused increases in both locomotor behavior and LFP activity in the VTA. Specifically, LFP activity was highest during the first 30 min following the cocaine injection and was most robust in Delta and Theta frequency bands; indicating the role of low frequency VTA activity in the initiation of acute stimulant-induced locomotor behavior. Our results suggest that LFP recording in freely moving animals can be used in the future to provide valuable information pertaining to drug induced changes in neural activity.

Endocrine disrupting chemical, bisphenol-A, induces breast cancer associated gene HOXB9 expression in vitro and in vivo.

HOXB9 is a homeobox-containing gene that plays a key role in mammary gland development and is associated with breast and other types of cancer. Here, we demonstrate that HOXB9 expression is transcriptionally regulated by estradiol (E2), in vitro and in vivo. We also demonstrate that the endocrine disrupting chemical bisphenol-A (BPA) induces HOXB9 expression in cultured human breast cancer cells (MCF7) as well as in vivo in the mammary glands of ovariectomized (OVX) rats. Luciferase assay showed that estrogen-response-elements (EREs) in the HOXB9 promoter are required for BPA-induced expression. Estrogen-receptors (ERs) and ER-co-regulators such as MLL-histone methylase (MLL3), histone acetylases, CBP/P300, bind to the HOXB9 promoter EREs in the presence of BPA, modify chromatin (histone methylation and acetylation) and lead to gene activation. In summary, our results demonstrate that BPA exposure, like estradiol, increases HOXB9 expression in breast cells both in vitro and in vivo through a mechanism that involves increased recruitment of transcription and chromatin modification factors.

Induction of deltaFosB in reward-related brain structures after chronic stress.

Acute and chronic stress differentially regulate immediate-early gene (IEG) expression in the brain. Although acute stress induces c-Fos and FosB, repeated exposure to stress desensitizes the c-Fos response, but FosB-like immunoreactivity remains high. Several other treatments differentially regulate IEG expression in a similar manner after acute versus chronic exposure. The form of FosB that persists after these chronic treatments has been identified as DeltaFosB, a splice variant of the fosB gene. This study was designed to determine whether the FosB form induced after chronic stress is also DeltaFosB and to map the brain regions and identify the cell populations that exhibit this effect. Western blotting, using an antibody that recognizes all Fos family members, revealed that acute restraint stress caused robust induction of c-Fos and full-length FosB, as well as a small induction of DeltaFosB, in the frontal cortex (fCTX) and nucleus accumbens (NAc). The induction of c-Fos (and to some extent full-length FosB) was desensitized after 10 d of restraint stress, at which point levels of DeltaFosB were high. A similar pattern was observed after chronic unpredictable stress. By use of immunohistochemistry, we found that chronic restraint stress induced DeltaFosB expression predominantly in the fCTX, NAc, and basolateral amygdala, with lower levels of induction seen elsewhere. These findings establish that chronic stress induces DeltaFosB in several discrete regions of the brain. Such induction could contribute to the long-term effects of stress on the brain.

Phospholipase Cgamma in distinct regions of the ventral tegmental area differentially modulates mood-related behaviors.

Neurotrophic factor signaling pathways modulate cellular and behavioral responses to drugs of abuse. In addition, chronic exposure to morphine increases expression of phospholipase Cgamma1 (PLCgamma1) (a protein involved in neurotrophic signaling) in the ventral tegmental area (VTA), a neural substrate for many drugs of abuse. Using viral-mediated gene transfer to locally alter the activity of PLCgamma1, we show that overexpression of PLCgamma1 in rostral portions of the VTA (R-VTA) results in increased morphine place preference, whereas PLCgamma1 overexpression in the caudal VTA (C-VTA) results in avoidance of morphine-paired compartments. In addition, overexpression of PLCgamma1 in R-VTA causes increased preference for sucrose and increased anxiety-like behavior but does not affect responses to stress or nociceptive stimuli. In contrast, overexpression of PLCgamma1 in C-VTA decreases preference for sucrose and increases sensitivity to stress and nociceptive stimuli, although there was a tendency for increased anxiety-like behavior as seen for the R-VTA. These results show that levels of PLCgamma1 in the VTA regulate responsiveness to drugs of abuse, natural rewards, and aversive stimuli and point to the possibility that distinct topographical regions within the VTA mediate generally positive versus negative responses to emotional stimuli. Moreover, these data also support a role for drug-induced elevations in PLCgamma1 expression in the VTA in mediating long-term adaptations to drugs of abuse and aversive stimuli.

Brain expression of pCREB in rats exposed to consummatory successive negative contrast.

A 32-to-4% sucrose devaluation leads to suppression of consummatory behavior relative to unshifted 4% sucrose controls. This is accompanied by an emotional response inducing memory consolidation. Expression levels of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB, a marker of synaptic plasticity) were higher after the first devaluation session than after the second in prelimbic cortex, anterior cingulate cortex, and dorso-medial striatum. The central nucleus of the amygdala showed a tendency to differential pCREB expression. This evidence contributes to identifying the brain circuit for one form of traumatic memory involving reward loss.

DeltaFosB: a molecular switch for long-term adaptation in the brain.

DeltaFosB is a unique transcription factor that plays an essential role in long-term adaptive changes in the brain associated with diverse conditions, such as drug addiction, Parkinson's disease, depression, and antidepressant treatment. It is induced in brain, in a region- and cell-type-specific manner by many types of chronic perturbations. Once induced, it persists for long periods of time due to its unusual stability. The transcriptional effects of DeltaFosB are complex, because the protein can function as both a transcriptional activator and repressor. Progress has been made in identifying specific target genes for DeltaFosB and in relating some of these genes to DeltaFosB's cellular and behavioral actions. Future studies will help us to better understand the biochemical basis of DeltaFosB's unique stability, as well as the precise molecular pathways through which this transcription factor produces its complex effects on neuronal plasticity and complex behavior.

Endogenous gonadal hormones modulate behavioral and neurochemical responses to acute and chronic cocaine administration.

Recent evidence demonstrates that there are sex differences in behavioral responses to cocaine. Further, reproductive gonadal hormones (estrogen, progesterone and testosterone) have been further implicated in mediating some of the cocaine-induced alterations. To better understand sex differences and the role of gonadal hormones in cocaine-induced locomotor and stereotypic behavior, intact and gonadectomized male and female Fischer rats were randomly assigned to either chronic cocaine (15 mg/kg) or saline treatments for 14 days followed by a challenge administration (7 days after the last cocaine/saline administration). Locomotor (ambulatory and rearing) and stereotypic activities were measured on days 1, 7 and 14 as well as after withdrawal/challenge with cocaine. Overall, intact female rats consistently showed a rapid (acquired by day 7) and longer lasting (persistent through the challenge dose) sensitization for all locomotor behaviors than any of the other groups. In contrast, intact males developed sensitization of these locomotor activities only in response to chronic cocaine administration, and after withdrawal and drug challenge the sensitization to cocaine-induced locomotor activity was no longer present. In female rats, gonadectomy affected ambulatory activity but not total and rearing responses after acute, sub-acute, chronic and challenge response to cocaine. On the other hand, castrated male rats were affected in cocaine-induced ambulatory activity but not rearing activity. In intact male rats, cocaine-induced stereotypic activity was rapidly and persistently sensitized after 7 days of cocaine administration, where gonadectomized male rats developed sensitization to cocaine-induced stereotypic activity only after a challenge cocaine administration. Although cocaine induced stereotypic activity, no statistically significant differences were observed between intact and ovariectomized female rats or throughout the different lengths of cocaine administration. After a challenge of cocaine, corticosterone levels were induced in all experimental groups. Moreover, no differences in levels of benzoylecgonine, a cocaine metabolite, were observed. Similar to our previous observations after acute cocaine administration, after challenge of cocaine, an increase in progesterone and a decrease in testosterone levels were observed in intact females and males, respectively. In summary, endogenous hormones seem to be involved in the behavioral activation and development of sensitization to cocaine.

Effects of cocaine on c-fos and preprodynorphin mRNA levels in intact and ovariectomized Fischer rats.

Psychostimulants such as cocaine have been shown to regulate c-fos and opioid gene expression in male rats. However, little information is available on cocaine effects in female rats or how the ovarian hormones, estrogen and progesterone, modulate these effects. In this study we used quantitative solution hybridization assays to measure c-fos and preprodynorphin (PDYN) mRNA levels after cocaine administration in the caudate/putamen of intact male and female rats or ovariectomized (OVX) female rats that were pretreated with vehicle, estrogen and/or progesterone. The c-fos mRNA levels were increased in intact male and female rats after 30min or 3h of one single cocaine injection and after 14 days of single daily cocaine injections. The c-fos mRNA levels were also increased after 30min of a single cocaine injection in OVX female rats that were treated with vehicle, estrogen and/or progesterone. The PDYN mRNA levels did not change after 30min, 3h or 14 days in intact male or female rats. However, PDYN mRNA levels were increased in the caudate/putamen of OVX female rats pretreated with vehicle or a combination of estrogen and progesterone but not in OVX female rats that were pretreated with either estrogen or progesterone alone. Our data suggest hormonal regulation of cocaine effects on PDYN mRNA levels which may modulate cocaine-induced behaviors in female rats.