Estrous Cycle Study Regarding Alcohol Use Disorder Through Endostatin Treatment

Sex and estrous cycle-linked differences in the effect of cannabidiol on panic-like responding in rats and mice

Estrogen receptor-β mRNA expression and estrogen positive neurons were found in the medial amygdaloid nuclei (ME), and neuronal nitric oxide synthase (nNOS) positive neurons were found in the amygdala in rats. We hypothesized that the expression of nNOS in the amygdala is associated with the estrogen level and exhibits periodicity during estrous cycle in female rats. 40 rats were divided into preoestrus, estrus, metoestrus, diestrus and control groups. The nNOS positive neurons were found in most amygdala regions. The ME contained a large number of densely stained and medium-sized (20~35μM) pyramidal cells. No nNOS positive structures were found in the control. The nNOS stain was stronger in the cytoplasm of neurons in preoestrus and metoestrus groups than in estrus and diestrus groups during the estrus cycle. The nNOS positive prominences were shorter in the ME than in other nuclei of the amgdala during whole estrous cycle. Most nNOS positive neurons were found in preoestrus groups (22.03±2.99), and some nNOS positive neurons were found in metoestrus groups (21.73±3.17), and few nNOS positive neurons were found in estrus groups (7.33±1.42) and diestrus groups (7.10±1.81). The number of nNOS positive neurons in preoestrus was higher than in estrus (p<0.01) and diestrus (p<0.01), and the number of nNOS positive neurons in metoestrus was higher than in estrus (p<0.01) and diestrus (p<0.01). The results indicate that the distribution of nNOS positive neurons in the ME varies with the estrogen level during whole estrus cycle. The estrogen level upregulates the amount of nNOS positive neurons in the ME. Interaction of estrogen and nitric oxide is involved in the neuroendocrine and sexual activity modulated partly by the amygdala.

The analgesic effect of neuropeptide S (NPS) in alcohol-dependent male and female rats.

Phrenic long-term facilitation (pLTF) and ventilatory long-term facilitation (vLTF) are well-defined models of respiratory neuroplasticity. In each model, acute intermittent hypoxia (AIH) stimulates persistent increases in respiratory neural drive or minute ventilation respectively. Recent findings from our laboratory indicate that sex hormones are necessary for the induction of respiratory neuroplasticity in both male and female rats. For example, AIH induced pLTF and vLTF only develop during the proestrus phase of the estrous cycle in female rats, notable for high levels of circulating 17β-estradiol, the most neuroactive form of estrogen. Removal of the ovaries (ovariectomy, OVX), the primary source of circulating sex hormones in females, also eliminates the development of pLTF and vLTF. Plasticity can be restored in OVX females with estradiol supplementation supporting a key role for estradiol signaling in development of respiratory plasticity. Testosterone, the principle sex hormone in males, is also important for development of respiratory plasticity. Male rats demonstrate reduced pLTF magnitude with aging, in correlation with reduced circulating testosterone levels, and removal of the gonads (gonadectomy, GDX) eliminates AIH-induced pLTF. Testosterone replacement is sufficient to restore pLTF in GDX male rats, but requires the activity of aromatase, the enzyme responsible for converting testosterone to estradiol, suggesting that estradiol may also play a key role in development of respiratory neuroplasticity in male rats. It is unknown whether conversion of testosterone to estradiol is necessary for development of AIH-induced plasticity in gonadally-intact male rats. To address this question, we used systemic injections of Letrozole, an aromatase inhibitor, as a first step towards determining the necessity of testosterone conversion to estradiol for development of AIH-induced respiratory neuroplasticity in gonadally-intact adult male rats. We hypothesized that aromatization of testosterone to estradiol would be necessary for induction of both AIH-induced vLTF and pLTF. Letrozole (1mg/kg), or a vehicle control, was administered subcutaneously over four days. On day three, vLTF was measured via whole-body plethysmography. On the fourth day, pLTF was quantified via phrenic nerve recordings. Researchers were blinded to treatment for all experiments and data analysis. Preliminary findings suggest that daily Letrozole injections impaired development of AIH-induced vLTF and pLTF, supporting our hypothesis. These data provide further evidence that estradiol plays a critical role in the development of respiratory neuroplasticity in both males and females.

The aim of the present study was to determine which of the anterior or posterior efferents of the suprachiasmatic nucleus (SCN) predominantly act for the maintenance of estrous cycle in female rats. A small knife (0.8 mm in radius) was used for anterior (ASD), posterior (PSD) and complete suprachiasmatic deafferentations (CSD). The rats subjected to CSD were divided into two groups, the first group bearing lesions on the medial preoptic nucleus (MPN; group CSD-MPN-L) and the other group bearing no lesions on the MPN (group CSD). For reference, anterior hypothalamic deafferentation (AHD) was carried out using a large knife (2.0 mm in radius). All rats with AHD or CSD-MPN-L and some of CSD rats showed persistent estrus, but all animals with ASD or PSD resumed regular estrous cycles after transient irregular periods. Ovaries of AHD, CSD and CSD-MPN-L rats contained very few, if any, corpora lutea, while the number of corpora lutea in ASD or PSD rats was not significantly different from that of sham-operated controls. Immunohistochemical examination revealed that in AHD rats LHRH immunoreactivity in the median eminence was markedly reduced as compared with that of sham-operated controls. In contrast, in ASD, PSD, CSD and CSD-MPN-L rats, the LHRH immunoreactivity in the median eminence was similar to that of sham-operated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Perceived Effectiveness of Medications Among Mental Health Service Users With and Without Alcohol Dependence

To evaluate whether intraganglionic calcitonin gene-related peptide induced differential migraine-like responses in male and female rats. Calcitonin gene-related peptide was injected in the trigeminal ganglion of male and female rats followed by assessment of periorbital mechanical allodynia with von Frey hairs. The influence of systemic treatment with sumatriptan or intraganglionic treatment with minocycline and propentofylline was determined on the calcitonin gene-related peptide-induced mechanical allodynia in male and female rats. One additional group was exposed to an aversive light 24 h after calcitonin gene-related peptide priming, followed by evaluation of periorbital mechanical threshold, and another group was tested in the elevated-plus maze. Intraganglionar calcitonin gene-related peptide-induced periorbital mechanical allodynia in female (0.5 to 6 h) and male rats (0.5 to 4 h). Systemic sumatriptan briefly attenuated the mechanical allodynia, but intraganglionar minocycline or propentofylline injection was effective only in male rats. Calcitonin gene-related peptide induced photic sensitivity in female and male rats (lasting 4 h and 1 h, respectively), as well as anxiety-like behavior. Intraganglionar calcitonin gene-related peptide may play a major role in migraine-like responses, including periorbital mechanical allodynia, light sensitivity and anxiety like-behavior. Female rats are likely to be more susceptible to calcitonin gene-related peptide effects and a better understanding of the sexual dimorphism in calcitonin gene-related peptide signaling may help to improve migraine therapy.

Alcohol use disorder (AUD) is a chronic psychiatric disorder characterized by escalated alcohol use and emergence of negative emotional states during withdrawal. Studies show a dysregulation of glucocorticoid signaling in AUD, affecting the hypothalamic-pituitary-adrenal (HPA) axis and the central amygdala (CeA). Excessive activation of glucocorticoid receptors (GRs) is hypothesized to promote cognitive dysfunction, negative emotional states, and escalated drinking. Previous work demonstrates a functional increase in GR activity in the CeA during alcohol withdrawal in alcohol-dependent animal models. Importantly, the GR antagonist mifepristone reduces alcohol-seeking and drinking behaviors in rodents and humans. The aims of this study were to determine differential CeA protein expression in alcohol-dependent rats and examine the effects of mifepristone treatment. We hypothesized that male and female rats will show sex differences in their CeA protein expression and that alcohol-induced reductions in brain anti-stress signaling (e.g., endocannabinoid system) will be normalized by mifepristone treatment. Male and female Wistar rats were exposed to chronic intermittent ethanol vapor (CIEV) or air (control) for 10 weeks. In week 7, half of each group received subcutaneous placebo pellets while the other half received mifepristone pellets (150mg, 21-day slow release). At the end of Week 10, the rats were sacrificed during acute (6-hour) CIEV withdrawal (and identical time in control groups). The CeA was analyzed using proteomics and ingenuity pathway analysis (IPA). In males, a total of 3050 proteins were detected in the CeA. Of these, 142 proteins were altered by alcohol (but not mifepristone), whereas an additional 253 proteins were altered by alcohol and “normalized” by mifepristone. In females, among the 2632 detected proteins, 46 were affected by alcohol (but not mifepristone), and 25 were altered by alcohol and restored by mifepristone. IPA revealed significant changes in canonical pathways, including the endocannabinoid neuronal synapse pathway and oxidative phosphorylation, affected by alcohol but normalized by mifepristone. Future studies will incorporate alcohol self-administration procedures in male and female rats to functionally examine proteins within these pathways as promising “druggable” targets that offer novel therapeutic avenues for AUD. Supported by NIH-NIAAA R01AA025996, P60AA009803, T32AA007577. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of acute ethanol (EtOH) on memory depend on several factors, including type of behavioral task. Sex differences in EtOH effects have been reported in humans and animals, and recognition memory can be influenced by circulating sex hormones. The aim of this study was to investigate the influence of sex and estrous cycle in the acute effects of EtOH on novel object recognition memory in rats. Male and female Wistar rats were part of one of the groups: control, 0.6-g/kg EtOH and 1.8-g/kg EtOH (administered intraperitoneally before the training session). The estrous cycle was evaluated by vaginal smear. The task was conducted in an open field arena. During training, animals were exposed to two identical objects, and test sessions were performed 1 h (short-term) and 24 h (long-term) later. One of the objects was changed in each test. Increased novel object exploration was shown by male and female controls in the short- and long-term tests, respectively. In the short-term test, females did not show preference for the novel object, and EtOH 1.8 g/kg impaired performance in males. In the long-term test, both sexes showed object discrimination, and 1.8-g/kg EtOH reduced preference for the new object in male rats. The phase of the cycle, the performance on proestrus was worse compared with other phases, and EtOH failed to impair performance mainly on estrous. In conclusion, while male rats displayed ethanol-induced recognition memory deficit, female rats were unaffected by EtOH impairing effects. In addition, the performance of female rats was influenced by the estrous cycle phases.

The nutritional status, prenatally and early postnatally, plays a critical role in postnatal growth and development. Early malnutrition may change the original programming of organs, especially those in developmental phases, which can result in long-term changes in metabolism. The association between a low birth weight and the increased risk on type 2 diabetes, hypertension and cardiovascular disease is well known. In the present study we investigated whether intrauterine malnutrition or direct postnatal food restriction affects the onset of puberty in male and female rats. Intrauterine growth retardation (IUGR) was induced by uterine artery ligation on day 17 of gestation and postnatal food restriction (FR) by litter-enlargement to 20 pups per mother from day 2 after birth until weaning (24 d). Both models of malnutrition resulted in a persistent growth failure postnatally. The parameter of the onset of puberty was balano-preputial-separation (BPS) in the male rat and vaginal opening (VO) in the female rat. In both male IUGR (n = 26) and FR (n = 20) rats, the age at BPS was significantly delayed, with 48.1 +/- 1.9 d (p < 0.0001) and 50.4 +/- 2.9 d (p < 0. 0001), respectively, compared with controls (n = 30) with 45.8 +/- 1.4 d. In female IUGR rats (n = 37) the age at VO was significantly delayed, with 37.4 +/- 2.7 d (p < 0.04) compared with 36.1 +/- 1.5 d in controls (n = 23), but not in female FR rats (n = 18) with 36.5 +/- 2.2 d. Weight at onset of puberty did not differ between male IUGR and control rats, 194.5 +/- 20.0 g and 201.7 +/- 16.8 g, respectively, but was significantly lower in male FR rats with a weight of 175.6 +/- 17.5 g (p < 0.0001). In female IUGR as well as in female FR rats, weight at onset of puberty was significantly lower compared with controls: weight in IUGR 106.1 +/- 13.1 g (p < 0.001), weight in FR 85.3 +/- 7.6 g (p < 0.0001) and weight in controls 116.9 +/- 9.3 g. We conclude that early malnutrition, during late gestation or direct postnatally, results in a delayed onset of puberty in IUGR and FR male rats and in IUGR female rats, but not in FR female rats. The onset of puberty in these growth retarded rats as well as in controls does not depend on the achievement of a certain, crucial weight. The perinatal period appears to be a "critical time period" for the maturational process of pubertal development.

To investigate the effects of one-week maternal separation (MS) on anxiety- and depression-like behaviors in adolescent and adulthood as well as adult hippocampal metabolomics simultaneously in offspring female and male rats. In the MS group, newborn SD rats were separated from their mothers for 3 h per day from postnatal days (PND) 2 to 8. The open field test (OFT), elevated plus mazes (EPM), novelty suppressed feeding test (NSFT), and forced swimming test (FST) were conducted during adolescence and adulthood. Serum corticosterone, mRNA expression of hippocampal inflammatory cytokines, and hippocampal untargeted metabolomics of offspring adult rats were examined using an assay kit, qRT-PCR, and UPLC-Q-TOF/MS. Both MS female and male rats showed similar behaviors in OFT, EPM, NSFT, and SPT, except for the latency to feeding during adolescence and the open arm entries during adulthood, showed statistical significance only in MS female rats. Serum corticosterone and hippocampal pro-inflammatory cytokines IFN-γ were significantly elevated in both female and male rats, and IL-1β and TNF-α were significantly increased only in female rats. In hippocampal metabolism, the identification of differential metabolites displayed 53 and 37 in female rats and male rats, respectively (with 35 common metabolites), which were involved in 33 and 30 metabolic pathways with 28 common pathways. One-week MS induced sex-specific anxiety- and depression-like behaviors in female and male offspring rats during adolescence and adulthood, as well as sex-differentiated characteristics in the hippocampus inflammatory cytokines and metabolomics of adult MS rats. From the experimental data, the effects of MS on the female offspring rats were more severe than those of the male offspring rats.

Restraint stress enhances alcohol intake in adolescent female rats but reduces alcohol intake in adolescent male and adult female rats.

Intermittent voluntary ethanol consumption combined with ethanol vapor exposure during adolescence increases drinking and alters other behaviors in adulthood in female and male rats.

PURPOSE It is well known that the eccentric muscle contraction (ECC) lead to more severe histological muscle damage than isometric muscle contraction (ISO). Recent studies indicated that exercise stimulates the expression of proteins related in apoptosis and increase apoptosis myonuclei. The purpose of this study was to examine the effect of ISO and ECC on the expression of apoptosis in male and female rats. METHODS Male and female wistar rats (13 weeks old) were used in the experiments. The left and right tibialis anterior muscles were subjected to 20 controlled ISO and ECC, respectively, by using an ankle extensor apparatus and surface electric stimulation under anesthetization. Rats of the same age were used as a normal control (CONT, n = 8). Induction of apoptosis was examined at one hour (1H), one day (1D), three days (3D) and seven days (7D) after ISO and ECC (n= 4–6, each group). Apoptosis was assessed by TUNEL assay, and labeled nuclei were identified under a fluorescence microscope. RESULTS Normal muscle fiber histology was found in CONT and ISO muscle in male and female rats. Following ECC, histological muscle damage such as focal inflammation and necrosis muscle fiber showed in male rats but not in female rats. In male and female rats, TUNEL-positive nuclei were significantly increased in ECC (male rats: 1H, 0.8 ±0.2; 1D, 1.2 ± 0.4; 3D, 3.1 ± 1.1; 7D, 2.8 ± 0.9. female rats: 1H, 0.3 ± 0.2; 1D, 0.8 ± 0.5; 3D, 1.4±0.2;7D, 1.3 ± 0.6, positive nuclei /mm2) compared with ISO (male rats: 1H, 0.2 ±0.1; 1D, 0.8 ± 0.2; 3D, 0.3 ±0.1; 7D, 0.6 ±0.3. female rats: 1H, 0.4 ±0.3; 1D, 0.2 ± 0.1; 3D, 0.1 ± 0.1; 7D, 0.2 ± 0.1). There were gender effects on ECC-induced apoptosis response (male >female; p <0.05). CONCLUSION These results suggest that ECC appear to cause greater apoptosis to skeletal muscle than ISO as well as histological muscle damage.

Pharmacokinetic significance of luminal multidrug and toxin extrusion 1 in chronic renal failure rats