Estrogen receptor beta (ERbeta) mRNA and protein in serotonin neurons of macaques.

This study used double in situ hybridization (ISH) to examine the colocalization of estrogen receptor beta (ERbeta) mRNA in serotonin neurons of rhesus macaques (Macaca mulatta). In addition, immunocytochemistry (ICC) was used to examine the expression and regulation of ERbeta protein in raphe neurons of the macaque midbrain. For double ISH, monkey specific riboprobes for ERbeta incorporating radiolabeled-UTP and a riboprobe for the human serotonin reuptake transporter (SERT) incorporating digoxigenin were applied to midbrain sections from spayed rhesus macaques. ERbeta mRNA hybridization signal was expressed in most cells containing SERT mRNA in the dorsal and median raphe and pons. There were also non-SERT neurons expressing ERbeta mRNA. In addition, ERbeta protein was detected with an affinity purified polyclonal antibody generated against a synthetic peptide corresponding to the D domain of human ERbeta conjugated to bovine serum albumin (provided by Dr. Philippa Saunders, MRC, Edinburgh). Midbrain sections containing the dorsal raphe from spayed rhesus macaques with and without hormone replacement therapy were processed for ERbeta immunostaining. ERbeta protein was detected at a similar intensity and in a similar number of cells in the dorsal raphe neurons in all treatment groups. Thus, the expression of ERbeta protein in the dorsal raphe was consistent with the expression of ERbeta mRNA. In conclusion, ERbeta mRNA is expressed by serotonin neurons and it is translated to protein. ERbeta protein, like ERbeta mRNA, is detected at similar levels in the presence or absence of ovarian hormones.     

Differences in hypothalamic serotonin between estrous phases and gender: an in vivo microdialysis study

The aim of the present study was to assess whether there are gender differences in (1) levels of extracellular serotonin (5-HT) in the forebrain, and (2) the effect on 5-HT of a reuptake inhibitor, paroxetine, or a releasing drug, fenfluramine. In vivo microdialysis was used to measure 5-HT in the hypothalamus of male and regularly cycling female rats. Hypothalamic 5-HT was significantly lower in estrous females (0.83±0.05 pg/sample, n=33) than in male rats (1.04±0.06 pg, n=38). Levels in diestrous females (0.98±0.09 pg, n=38) were not significantly different from males. Paroxetine (1 mg/kg) increased hypothalamic 5-HT in males, and diestrous and estrous females to 2 pg/sample. However, the increase in hypothalamic 5-HT produced by a maximally effective dose of paroxetine (10 mg/kg) was significantly greater in male rats and during diestrous than during estrous.

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-Fenfluramine (10 mg/kg) evoked an increase in extracellular 5-HT to 15 pg/sample in all groups. A higher dose of

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-fenfluramine (20 mg/kg) produced a significantly greater increase in hypothalamic 5-HT in males than in females during estrous or diestrous. These results are consistent with other evidence that during estrous, when rats are responding to peak levels of estrogen and progesterone, 5-HT release is decreased.     

Selective estrogen receptor-beta (SERM-beta) compounds modulate raphe nuclei tryptophan hydroxylase-1 (TPH-1) mRNA expression and cause antidepressant-like effects in the forced swim test

Estrogen acts through two molecularly distinct receptors termed estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) which bind estradiol with similar affinities and mediate the effects of estrogen throughout the body. ERα plays a major role in reproductive physiology and behavior, and mediates classic estrogen signaling in such tissues as the uterus, mammary gland, and skeleton. ERβ, however, modulates estrogen signaling in the ovary, the immune system, prostate, gastrointestinal tract, and hypothalamus, and there is some evidence that ERβ can regulate ERα activity. Moreover, ERβ knockout studies and receptor distribution analyses in the CNS suggest that this receptor may play a role in the modulation of mood and cognition. In recent years several ERβ-specific compounds (selective estrogen receptor beta modulators; SERM-beta) have become available, and research suggests potential utility of these compounds in menopausal symptom relief, breast cancer prevention, diseases that have an inflammatory component, osteoporosis, cardiovascular disease, and inflammatory bowel disease, as well as modulation of mood, and anxiety. Here we demonstrate an antidepressant-like effect obtained using two SERM-beta compounds, SERM-beta1 and SERM-beta2. These compounds exhibit full agonist activity at ERβ in a cell based estrogen response element (ERE) transactivation assay. SERM-beta1 and 2 are non-proliferative with respect to breast as determined using the MCF-7 breast cancer cell-based assay and non-proliferative in the uterus as determined by assessing the effects of SERM-beta compounds on immature rat uterine weight and murine uterine weight. In vivo SERM-beta1 and 2 are brain penetrant and display dose dependent efficacy in the murine dorsal raphe assays for induction of tryptophan hydroxylase mRNA and progesterone receptor protein. These compounds show activity in the murine forced swim test and promote hippocampal neurogenesis acutely in rats. Taken together these data suggest that ERβ may play an important role in modulating mood and the ERβ specific compounds described herein will be useful tools for probing the utility of an ERβ agonist for treating neuroendocrine-related mood disturbance and menopausal symptoms.     

Systemic uptake inhibition decreases serotonin release via somatodendritic autoreceptor activation

In vivo microdialysis was used to examine the effects of peripheral uptake inhibition on extracellular serotonin (5-HT). Previous results from this lab indicated that systemic fluoxetine caused a decrease in 5-HT when terminal uptake was inhibited by local infusion of the uptake blocker. We hypothesized that the decrease in 5-HT levels in the terminal region was due to an increase in 5-HT in the vicinity of the inhibitory somatodendritic autoreceptors in the dorsal raphe nucleus (DRN). To test this prediction, rats were implanted with probes in both the basal diencephalon (a nerve terminal region) and the DRN (the cell body region). Fluoxetine (10 mg/kg i.p.) increased extracellular 5-HT, in a depolarization-dependent manner, by approximately 140% in both areas. In a separate experiment, fluoxetine was infused into the diencephalon overnight to block nerve terminal uptake sites. This pretreatment caused an eight- to 10-fold increase in 5-HT levels. Subsequent systemic fluoxetine, sertraline, or paroxetine, produced a 50% decrease in extracellular 5-HT in the diencephalon, presumably due to activation of the 5-HT_1A somatodendritic autoreceptors. Consistent with this hypothesis, systemic administration of the 5-HT₁ antagonists spiperone, penbutolol, or WAY100135 reversed the fluoxetine-induced decrease in 5-HT to approximately 85% of the pre-fluoxetine baseline levels. Likewise, pretreatment with penbutolol, but not selective ß-adrenergic antagonists, blocked the fluoxetine-induced decrease in release. These findings suggest that the ability of acute systemic 5-HT uptake inhibition to elevate nerve terminal 5-HT is limited by autoreceptor activation following elevation of 5-HT in the DRN. © 1995 Wiley-Liss, Inc.     

Diverse actions of ovarian steroids in the serotonin neural system

All of the serotonin-producing neurons of the mammalian brain are located in 10 nuclei in the mid- and hindbrain regions. The cells of the rostal nuclei project to almost every area of the forebrain and regulate diverse neural processes from higher order functions in the prefrontal cortex such as integrative cognition and memory, to limbic system control of arousal and mood, to diencephalic functions such as pituitary hormone secretion, satiety, and sexual behavior. The more caudal serotonin neurons project to the spinal cord and interact with numerous autonomic and sensory systems. All of these neural functions are sensitive to the presence or absence of the ovarian hormones, estrogen and progesterone. We have shown that serotonin neurons in nonhuman primates contain estrogen receptor beta and progestin receptors. Thus, they are targets for ovarian steroids which in turn modify gene expression. Any change in serotoninergic neural function could be manifested by a change in any of the projection target systems and in this manner, serotonin neurons integrate steroid hormone information and partially transduce their action in the CNS. This article reviews the work conducted in this laboratory on the actions of estrogens and progestins in the serotonin neural system of nonhuman primates. Comparisons to results obtained in other laboratory animal models are made when available and limited clinical data are referenced. The ability of estrogens and progestins to alter the function of the serotonin neural system at various levels provides a cellular mechanism whereby ovarian hormones can impact cognition, mood or arousal, hormone secretion, pain, and other neural circuits.     

Cardiovascular Reactivity During Positive and Negative Marital Interactions

Marriage reduces risk of cardiovascular disease (CVD) but marital stress increases risk, perhaps through cardiovascular reactivity (CVR). However, previous studies have lacked controls necessary to conclude definitively that negative marital interactions evoke heightened CVR. To test the specific effects of marital stress on CVR, 114 couples engaged in positive, neutral, or negative interactions in which speaking and task involvement were controlled. Compared to positive and neutral conditions, negative discussions evoked larger increases in systolic blood pressure, heart rate, and cardiac output, and larger decreases in peripheral resistance and pre-ejection period--similarly for men and women. Hence, CVR could contribute to the effects of marital difficulties on CVD. Previous evidence of sex differences in this effect might reflect factors other than simple reactivity to negative interactions.     

Estrogen receptor-beta regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe.

BACKGROUND: Distinct expression patterns of estrogen receptor (ER)-alpha and ER-beta are displayed in the murine central nervous system. ER-beta is the predominant form of the receptor expressed in the murine midbrain dorsal raphe nucleus (DRN). Tryptophan hydroxylase (TPH) is abundantly expressed in the serotonergic neurons of the DRN and is regulated by estrogen in both the monkey and the guinea pig. METHODS: In this study we used immunocytochemistry to show that ER-beta and TPH are colocalized in the serotonergic cells of the murine DRN. We utilized the ER-alpha and ER-beta gene deletion mouse models and in situ hybridization to demonstrate that ER-beta is responsible for regulating TPH1 mRNA expression. RESULTS: Estrogen increased TPH1 mRNA expression in the DRN of wild type and ER-alpha knockout mice (alpha-ERKO) but not ER-beta knockouts (beta-ERKO). CONCLUSIONS: These data indicate that ER-beta is responsible for mediating estrogen regulated TPH1 expression in the murine DRN.     

Evaluating obesity in fibromyalgia: neuroendocrine biomarkers, symptoms, and functions

The aim of this study was to investigate the associations between obesity and fibromyalgia syndrome (FMS). This study was conducted at the University of Utah Pain Management and Research Center, Salt Lake City, Utah. Thirty-eight FMS patients were included in this study. Neuroendocrine indices (catecholamines, cortisol, C-reactive protein [CRP], and interleukin-6), symptom measures (Fibromyalgia Impact Questionnaire), sleep indices (Actigraph), and physical functioning (treadmill testing) were measured. Body mass index (BMI) provided the primary indicator of obesity. Approximately 50% of the patients were obese and an additional 21% were overweight. Strong positive associations were found between BMI and levels of IL-6 (r = 0.52) and epinephrine (r = 0.54), and somewhat weaker associations with cortisol (r = 0.32) and CRP (r = 0.37). BMI was also related to maximal heart rate (r = 0.33) and inversely related to distance walked (r = −0.41). BMI was associated with disturbed sleep: total sleep time (r = −0.56) and sleep efficiency (r = −0.44). No associations between self-reported symptoms and BMI were found. This study provides preliminary evidence suggesting that obesity plays a role in FMS-related dysfunction.