Intact Cycling Females Research Articles

Cues play a critical role in estrous cycle-dependent enhancement of cocaine reinforcement.

While preclinical work has aimed to outline the neural mechanisms of drug addiction, it has overwhelmingly focused on male subjects. There has been a push in recent years to incorporate females into existing addiction models; however, males and females often have different behavioral strategies, making it important to not only include females, but to develop models that assess the factors that comprise female drug addiction. Traditional self-administration models often include light or tone cues that serve as discriminative stimuli and/or consequent stimuli, making it nearly impossible to disentangle the effects of cue learning, the cues themselves, and acute effects of psychostimulant drugs. To disentangle the interaction between drug-associated cues and the consummatory and appetitive responding driven by cocaine, we have developed a new behavioral procedure that combines Pavlovian-instrumental transfer with behavioral economic analysis. This task can be completed within a single session, allowing for studies looking at estrous cycle stage-dependent effects in intact cycling females, something that has been difficult in the past. In this study, we found no differences in self-administration across the estrous cycle in the absence of cues; however, when cues were introduced, the cues that acquired value during estrus-but not during diestrus or in males-increased motivation. Cues paired during estrus also increased c-fos expression to a greater extent in striatal regions, an effect that may underlie the observed increases in seeking induced by these cues, even weeks later. Together, these data suggest that fundamental differences in the motivational properties of psychostimulant drugs between males and females are complex and are driven primarily by the interaction between drug-associated stimuli and drug effects.

The role of estradiol on social stress induced depressive‐like behavior and neuroinflammation in females

It has long been established that repeated exposure to social stress can lead to the development of psychosocial disorders, such as depression. Strikingly, women are more sensitive to stress‐related disorders, such as depression compared to men. This enhanced stress sensitivity in females begins at the onset of puberty and ends during menopause, suggesting that ovarian hormones could be a large contributing factor to this phenomenon. However, the mechanism by which ovarian hormones could be driving this enhanced stress sensitivity is unknown. Using a modified social defeat paradigm which consists of a rat bearing witness to an aggressive social defeat encounter, we have previously shown that witness stress produces depressive‐like anhedonia and enhanced pressor and tachycardic responses selectively in intact cycling females while females that are ovariectomized (OVX) are resistant, making this an ideal model to study mechanisms of stress susceptibility in females. We recently demonstrated that neuroinflammation is crucial in the development of depressive‐like anhedonia in socially stressed males. The purpose of this study was to determine the effects of estradiol on social stress‐induced neuroinflammation within the central amygdala (CeA) and locus coeruleus (LC) of witness stressed females. Study 1 determined the differential neuroinflammatory consequences of witness stress in intact cycling vs. OVX females. Much like our behavioral data suggests, intact cycling females show enhanced neuroinflammation in the CeA compared to OVX females. Interestingly, neuroinflammation was not elevated in the LC in response to witness stress, but rather OVX females demonstrated stress‐induced reductions of IL‐1β within the LC vs. controls. In order to determine the discrete role of estradiol in these neuroinflammatory responses to stress, study 2 determined cytokine levels in OVX females that received estradiol or placebo replacement. While estradiol replacement did not result in the emergence of stress‐induced depressive‐like anhedonia, it did promote anxiety‐like behavior as evidenced by increased burying during witness stress exposure. Based on this enhanced anxiety‐like response, we predict concomitant increases in neuroinflammation within the CeA, a brain region known to contribute to behavioral stress responses. Therefore, the current study suggests that ovarian hormones, potentially estrogen, may mediate enhanced stress sensitivity in females by promoting stress‐induced inflammation in critical brain regions involved in the behavioral stress response, providing novel therapeutic targets to study female stress resiliency and susceptibility.Support or Funding InformationASPIRE18080‐16‐41861, 15SDG22430017, and P20GM103641

Prolame ameliorates anxiety and spatial learning and memory impairment induced by ovariectomy in rats

N-(3-hydroxy-1, 3, 5 (10) estratrien-17beta-yl)-3-hydroxypropylamine (17β aminoestrogen, prolame) is a steroidal compound with weak estrogen-related trophic-proliferative effects in uterus. Contrasting with 17β-estradiol (E2) pro-coagulant effects, this compound has high anticoagulant and antiplatelet effects. It has been extensively demonstrated that E2 plays important roles in brain function. However, prolame's influence on central nervous system has not been documented. In this study, we evaluated the effects of prolame replacement in young ovariectomized rats on spatial learning and memory and anxiety, correlating pyramidal cell dendritic spine density changes and neuronal nitric oxide synthase (nNOS) expression in the hippocampus. Ovariectomized young rats were treated with prolame for 4weeks. Three other groups were used as physiological, pathological, and pharmacological references as follow: gonadally intact cycling females, ovariectomized, and ovariectomized with 17β-estradiol treatment respectively, for the same time period. Experiment 1 investigated the behavioral effects of prolame on anxiety and spatial learning using elevated plus maze (EPM) and Morris water maze (MWM) paradigms respectively. Experiment 2 studied the dendritic spine density and neuronal nitric oxide synthase expression in the hippocampus of the 4 experimental groups. Similar to estradiol, prolame reversed the anxiogenic effects of ovariectomy, evaluated by EPM, and enhanced MWM performance to the level of gonadally intact subjects. Hippocampi from prolame-treated rats exhibited enhanced nNOS immunoreactivity and its relocation in dendritic compartments, as well as recovery of dendritic spine density loss in pyramidal neurons. Hence, prolame may provide an alternative option for ameliorating neurological symptoms caused by surgical menopause.

The Role of Oestradiol in Sexually Dimorphic Hypothalamic‐Pituitary‐Adrena Axis Responses to Intracerebroventricular Ethanol Administration in the Rat

Systemic ethanol (EtOH) administration activates the hypothalamic-pituitary-adrenal (HPA) axis of rats in a sexually dimorphic manner. The present studies tested the role played by the central nervous system (CNS) in this phenomenon. To localise the effects of the drug to the brain, we utilised an experimental paradigm whereby a small, nontoxic amount of the drug was delivered via intracerebroventricular (i.c.v.) injection. EtoH administered i.c.v. rapidly diffuses throughout the cerebrospinal fluid and brain, and does not cause neuronal damage or have any long-term physiological or behavioural effects. Experimental groups included intact males, intact cycling females, and ovariectomised (OVX) animals with or without replacement oestradiol (E(2)). Intracerebroventricular EtOH-induced HPA hormonal activation was determined by measuring plasma adrenocorticotrophin (ACTH) levels. Activation of brain areas that both regulate HPA function and are responsive to gonadal hormones was determined using expression of the transcription factor c-fos (Fos) as a marker of neuronal activity. We observed sex- and oestrous cycle- dependent differences in HPA activation by EtOH as measured by both these parameters. ACTH secretion was highest in females in pro-oestrus or oestrus, just prior to or after the endogenous peak of E(2), as was Fos expression in the paraventricular nucleus of the hypothalamus (PVN) and the locus coreuleus (LC) of the brainstem. In OVX animals, E(2) replacement caused an increase in PVN and LC Fos expression in response to i.c.v. EtOH compared to OVX controls, but a decrease in ACTH secretion. Taken together, these results indicate that at the level of the CNS, EtOH stimulates HPA activity more robustly at times when the effects of E(2) are high, but that E(2) alone is not responsible for this effect. The data further suggest that the LC plays an important role in the circuitry, which appears to be different from that activated following the systemic administration of EtOH.

Up-regulation of pituitary [125I]insulin-like growth factor-I (IGF-I) binding and IGF binding protein-2 and IGF-I gene expression by estrogen.

To evaluate the effects of estrogen on the rat pituitary insulin-like growth factor (IGF) system, binding of [125I]IGF-I and in situ hybridization for IGF-I, the IGF-I receptor and IGF binding protein-2 (IGFBP-2) were coupled with quantitative autoradiography. The groups included intact cycling females, intact males, and gonadectomized males and females with or without estrogen pellet implants. Binding of [125I]IGF-I in the anterior lobe of the pituitary occurred in dense clusters over a diffuse lower density background. [125I]IGF-I binding was significantly increased in the estrogen-treated groups and was highest at proestrus compared to the rest of the estrous cycle. IGF-I receptor messenger RNA (mRNA) was distributed diffusely through the anterior pituitary and was not different between the respective gonadectomized and estrogen-treated groups. IGFBP-2 mRNA was clustered throughout the anterior pituitary and was significantly higher in the estrogen-treated groups as noted above for [125I]IGF-I binding. IGF-I mRNA was diffuse throughout the anterior pituitary and was also significantly higher in the estrogen-treated groups. In the neural lobe, [125I]IGF-I binding, IGFBP-2 mRNA, IGF-I receptor mRNA, and IGF-I mRNA were all uniformly distributed and did not differ between groups. The results show that circulating estrogen differentially regulates components of the pituitary IGF-I system in a region-specific manner and suggest that a portion of IGF binding in the anterior pituitary may be to IGFBP-2.

Up-regulation of pituitary [125I]insulin-like growth factor-I (IGF-I) binding and IGF binding protein-2 and IGF-I gene expression by estrogen

To evaluate the effects of estrogen on the rat pituitary insulin-like growth factor (IGF) system, binding of [125I]IGF-I and in situ hybridization for IGF-I, the IGF-I receptor and IGF binding protein-2 (IGFBP-2) were coupled with quantitative autoradiography. The groups included intact cycling females, intact males, and gonadectomized males and females with or without estrogen pellet implants. Binding of [125I]IGF-I in the anterior lobe of the pituitary occurred in dense clusters over a diffuse lower density background. [125I]IGF-I binding was significantly increased in the estrogen-treated groups and was highest at proestrus compared to the rest of the estrous cycle. IGF-I receptor messenger RNA (mRNA) was distributed diffusely through the anterior pituitary and was not different between the respective gonadectomized and estrogen-treated groups. IGFBP-2 mRNA was clustered throughout the anterior pituitary and was significantly higher in the estrogen-treated groups as noted above for [125I]IGF-I binding. IGF-I mRNA was diffuse throughout the anterior pituitary and was also significantly higher in the estrogen-treated groups. In the neural lobe, [125I]IGF-I binding, IGFBP-2 mRNA, IGF-I receptor mRNA, and IGF-I mRNA were all uniformly distributed and did not differ between groups. The results show that circulating estrogen differentially regulates components of the pituitary IGF-I system in a region-specific manner and suggest that a portion of IGF binding in the anterior pituitary may be to IGFBP-2.

Divergent Regulation of Gonadotropin Subunit mRNA Levels by Androgens in the Female Rat1

To examine the effects of gonadal steroids on the pretranslational regulation of the gonadotropin subunits in the female, adult female rats, beginning 7 or 28 days after ovariectomy, received daily injections of testosterone propionate (T), dihydrotestosterone propionate (D), or estradiol benzoate (E) for 7 days. Intact cycling females and ovariectomized rats that received vehicle served as controls. Serum was obtained for LH and FSH levels to assess changes in gonadotropin secretion. Total RNA from individual rats was recovered and analyzed by blot hybridization with specific radiolabeled cDNA probes for the alpha, LH beta, and FSH beta subunits. Autoradiographic bands were quantitated and standardized to mRNA levels in the intact animals. Ovariectomy resulted in a rise in serum gonadotropin levels and all three gonadotropin subunit mRNA levels. Estrogen replacement resulted in suppression of alpha, LH beta, and FSH beta mRNAs whether given at 7 or 28 days after ovariectomy. In contrast, whereas androgen replacement decreased alpha and LH beta mRNAs, D or T did not consistently suppress FSH beta mRNAs. We conclude that chronic estrogen administration to the castrated female rat uniformly suppresses all three gonadotropin subunit mRNA levels. In female rats, as in male rats, chronic androgen administration fails to negatively regulate FSH beta mRNAs.

Dual estradiol action in diencephalon and the regulation of sociosexual behavior in female golden hamsters

Previously, we found that single implants of estradiol (E2) placed in the ventromedial hypothalamus (VMH) but not the anterior hypothalamus (AH) facilitated precopulatory, i.e. vaginal scent marking, and copulatory, i.e. lordosis, behavior following progesterone administration. However, the duration of lordosis was markedly attenuated in comparison to the duration shown by intact cycling females. This study was designed to examine whether dual implantation of E2 in the diencephalon would facilitate patterns of precopulatory and copulatory behaviors similar to those shown by intact cycling females. One E2 implant was placed in either the medial preoptic area (MPO) or AH and a second E2 implant was placed in the VMH. Control females were tested following E2 application at only the MPO or AH region in conjunction with a cholesterol implant in the VMH. An additional control group was tested with females implanted with cholesterol at both MPO-AH regions and the VMH. During a 2-day postimplantation test period, vaginal marking scores were elevated for both single and dual E2 implanted females and agonistic response patterns toward males declined significantly. In addition, a significant inverse relationship was found between the number of vaginal marks and bites exhibited by females with single E2 implants in the MPO, whereas these two response patterns were positively correlated in females with E2 stimulation occurring only in the AH region. No significant relationship was found between vaginal marking and biting attack for females receiving dual E2 stimulation. Systemic progesterone administration on the third postimplantation day facilitated sexual receptivity in the majority of females with dual E2 implants (greater than 90%). These receptive females displayed lordotic responsiveness that closely matches the full display of sexual receptivity shown by intact cycling females. In contrast, only one female with a single E2 implant in the AH region showed sexual responsiveness. The results demonstrate that: precopulatory vaginal marking and biting attack are mediated by E2 action in the MPO and AH but in a different manner; additional action of E2 in the VMH diminishes the distinctive precopulatory behavioral effects of E2 in the MPO and AH suggesting an influential role of the VMH in regulating sociosexual activities; and E2 action in either the MPO or AH region in conjunction with E2 action in the VMH may be necessary in order to facilitate the species-typical display of lordotic responsiveness.

Antisera to porcine follicular fluid in monkeys: neutralization of human and pig inhibin activity in vivo and in vitro.

Repetitious administration of charcoal-stripped porcine follicular fluid (pFF) to four intact cycling females and six long term castrate rhesus monkeys was associated with a gradual onset of resistence to the FSH-suppressing effects of pFF. After a treatment hiatus of 6 or more months, the pFFinduced selective suppression of FSH in circulation was again demonstrable, but only transiently; that is, within 10–14 weeks after the resumption of therapy, even large doses of pFF were no longer effective in reducing serum FSH in these monkeys. One monkey was studied through seven regimens over 4 yr. Accordingly, our series of investigations was divided into three parts. First, we observed that these changing FSH patterns were apparently due to cyclic fluctuations of pFF antibodies in conjunction with the intermittent schedule of pFF administration. The amnestic response to booster injections occurred within 7–14 days, followed by a gradual decline in the anti-pFF titer during succeeding months. Second, we tested whether these anti-pFF sera would neutralize inhibin activities (selective FSH suppression) in rat pituitary cell cultures. Indeed, the anti-pFF sera were effective in blocking the FSH-suppressing activity of pFF. Again, each successive episode of pFF administration was correlated with an enhancement of the anti-pFF titer, indicated by greater potency of the monkey antisera in the in vitro setting. Thirdly, we assessed whether the monkey's anti-pFF sera would neutralize the inhibin activity in human FF (hFF); thereby establishing cross-reactivity with human inhibin. Clearly, the mixture of anti-pFF serum along with charcoal-stripped hFF prevented the selective suppression of FSH secretion that occurred when control monkey serum was used instead. These findings indicate that porcine inhibin present in charcoalstripped pFF is highly immunogenic in monkeys; further, some of the antibodies generated cross-react with the active inhibin component in hFF. Indeed, the neutralization of inhibin activity in hFF by anti-pFF monkey sera suggests some significant structural similarities between human and pig inhibins; yet, concurrently, the ready immunogenicity of pFF administered to monkeys indicates clear recognition of the antigen as an intrinsically foreign protein. These anti-pFF monkey sera may be useful in the establishment of RIA for porcine and/or human inhibins as well as the preparation of improved reagents for studying the physiological significance of this substance (s) in follicular fluid and circulation.