Changes in estrogen are correlated with a variety of effects in the CNS, such as changes in pain transmission, headache, dizziness, nausea, temperature regulation, and mood 41. Serotonin systems regulate these same functions4143 in a direction consistent with mediation of E2 effects. For pain, E2 acts as a central analgesic,44 and pain sensation is inhibited by the activation of some serotonergic neurons 4. Analgesic drugs that exploit this effect at the 5HT_2A receptor are already available 445464748. We hypothesize that E2's upregulation of the 5HT_2A receptor in the brain might contribute to E2-mediated pain relief, in which case central administration of 5HT_2A receptor antagonists would decrease E2's analgesic effects. In the spinal cord, altered expression of 5HT_2A receptors can both increase and decrease pain 4849. E2's upregulation of 5HT_2A in the spinal cord could be a factor in the development of fibromyalgia, which presents as increased generalized pain sensation. Serotonergic regulation of fibromyalgia is supported by evidence that fibromyalgia is comorbid with other serotonin-related pathologies,50 and that fibromyalgia patients have altered tryptophan metabolism51 and can be treated with 5HT_2A antagonists 50. E2's effect on serotonin could also explain why fibromyalgia is more frequently observed in females than males 52.

Females are also at greater risk for headaches,43 which can result from vasodilation in the brain 53. Activation of an additional serotonin receptor, 5HT_1B, is one mechanism by which vasodilation occurs. 5HT_1B receptors are not uncoupled by E2 (unlike 5HT_1A receptors), and their vasodilatory effect is typically balanced by activation of 5HT_2A receptors, which result in vasoconstriction 54. After E2 exposure, increased serotonin concentrations result in greater activation of both the 5HT_1B and 5HT_2A receptors. Under normal conditions, upregulation and activation of 5HT_2A receptors enable them to balance the effects of 5HT_1B receptors 272855. We suggest that females' increased headache risk might result if high serotonin concentrations are maintained without adequate compensatory 5HT_2A activity.

Two of the major side effects of E2 treatment are dizziness and nausea, which are controlled in the CNS. The mechanism by which these side effects occur has not been fully elucidated. It is possible that E2's effect on serotonin pathways is responsible for these symptoms, as 5HT_2A receptors activate vestibular neurons (which maintain balance)56 and are found in emetic centers, which are involved in chemically-induced vomiting 57. Our hypothesis is corroborated by the use of serotonergic drugs to minimize these side effects of E2 treatment 58.

The loss of estrogen at menopause results in decreased density of 5HT_2A receptors and lower activity of serotonin, which could explain aberrant temperature regulation, including hot flashes and night sweats. Although the effects of temperature changes are felt throughout the body, 5HT_2A receptors in the CNS are responsible for temperature regulation. Administration of drugs acting at the 5HT_2A receptor restores normal temperature regulation following ovariectomy59 and chemically induced changes in body temperature60 The nighttime prevalence of hot flashes and night sweats could be a result of the conversion of serotonin to melatonin at night, resulting in lower circulating serotonin levels 61. Phytoestrogens preferentially bind to ERβ receptors30 and are effective at reducing hot flashes and night sweats 62. The mechanism by which these compounds work could be an ERβ-produced upregulation of 5HT_2A receptors.

Depression is more common in women than in men and is known to be mediated by serotonin receptor levels 4363. Specifically, depression is linked to decreased density of serotonin receptors and decreased efficacy of serotonin in the brain. The increased risk, timing of onset, and effectiveness of treatment of depression in women may be mediated by estrogen's effect on serotonin receptors. The onset of depression in women is a characteristic of times when estrogen levels are relatively low (in early pregnancy, postpartum, and around and following menopause) or low in comparison to progesterone (the luteal phase of the menstrual cycle) 6465. In women with depression around or following menopause, the effectiveness of treatment with selective serotonin reuptake inhibitors (SSRIs) is enhanced by simultaneous administration of estrogen,63 and doses of estrogen alone are effective at treating premenstrual, postpartum, and perimenopausal depression, especially for depression linked to aberrant expression of 5HT_2A receptors 2566. ERβ regulates the antidepressant effect of E2 in mice; ERβ knockout mice fail to show the decrease in immobility usually induced by E2 doses in a forced swim test 67. The increased levels of serotonin and increased activity of the 5HT_2A receptor caused by E2 could be the mechanism for E2's antidepressant effects, in which case 5HT_2A receptor agonists could also enhance the anti-depressant effects of E2.

Estrogen and serotonin also affect the skeletal system. As bones grow, they are continually remodeled and reshaped. Normal bone development is affected by growth hormone, parathyroid hormone, calcitonin, and environmental factors like dietary calcium intake and physical activity. In addition to these factors, estrogen and serotonin play an important role in the development and maintenance of bone mass. For bone growth to occur, two types of cells are required: osteoblasts, which form new bone, and osteoclasts, which resorb bone. During puberty, osteoclasts and osteoblasts are in balance and resorb and build bone simultaneously, but osteoporosis results when osteoclasts increase relative to osteoblasts. These effects have been linked to E2 concentrations in both males and females,6869 and we propose that they can be explained by examining E2-produced changes in serotonergic function in bone growth and loss. 5HT_2A receptor activation causes an increase in expression of osteoblast progenitor cells, maintaining bone density 70. SERT activation, in contrast, increases osteoclasts in bone, aiding in bone growth in childhood,71 but resulting in loss of bone density and increases in extracellular Ca⁺⁺ postpartum7273 and in menopause 7475. Studies of female mice lacking the ERα, the ERβ, or both suggest these two receptors might counterbalance each other's effects on longitudinal bone growth,76 with ERβ primarily responsible for decreasing bone growth and increasing bone resorption 77. Because ERα and ERβ have opposing effects on serotonin systems, we hypothesize that mediation by serotonin could explain E2's effects on the skeletal system: the decrease in bone density observed following menopause or when E2 function is otherwise compromised. However, bone loss begins around age 30 in men and women and this early bone loss cannot be entirely explained by differences in E2 concentrations or by our proposed model 78.

In the vascular system, estrogen and serotonin have been shown to individually alter clotting, cholesterol, vasoconstriction, and heart attacks. Both high and low levels of E2 have been associated with increased risk of thromboembolism; high levels result in increased clot formation, while low levels result in slower clot breakdown. Unusually high concentrations of estrogen (beyond normal physiological levels) directly increase the likelihood of clotting by increasing production of clotting factors VII through X in the liver 41. In addition, these levels of E2 might increase clotting by increasing serotonin, which is constitutively present in human plasma and platelets and works to promote clotting679 and increase density of platelets 58. Increased clotting and thromboembolism at low concentrations of E2 80 can also be explained using serotonergic changes. Postmenopausal women have longer latency to lysis of clots, and E2 replacement therapy returns latencies to pre-menopausal levels 81. Patients with slower clot breakdowns have decreased uptake and release of serotonin from platelets,82 and at low E2 levels serotonin's ability to break down clots via the 5HT_2A receptor is limited,8384 so we suggest that lower serotonin activity associated with lower E2 levels could also contribute to increased clotting.

Increased concentrations of E2 are also associated with decreased cholesterol, and at menopause, there is an increase in total serum cholesterol, which is reduced by estrogen-containing hormone replacement therapy 85. We suggest higher cholesterol after menopause is linked to the effects of serotonin. Serotonin increases membrane fluidity by incorporation of cholesterol into membranes, decreasing bioavailable cholesterol 8687. Increased membrane fluidity also increases serotonergic function, creating a positive feedback loop 8889. If serotonin is an intermediary between estrogen and cholesterol, then in the presence of high concentrations of E2, we would expect more cholesterol incorporated into membranes, thereby reducing cholesterol present in the plasma. Our hypothesis would be supported if the administration of drugs that reduce concentrations of serotonin in the plasma cause increases in plasma cholesterol despite consistent levels of E2.

Both clotting and cholesterol contribute to heart attack risk. Women are at lower risk of heart attack than men prior to menopause, but changes in the vascular system after menopause result in the loss of protection from heart disease 4143. In females, recent evidence suggests that physiological levels of E2 protect against heart attacks, while testosterone makes heart attacks more likely 90. E2 acting at ERβ is responsible for this protective effect, as mice lacking ERβ have greater mortality and increased heart failure indicators following experimentally induced myocardial infarctions 91. We hypothesize that these effects in females can be explained in part by serotonin receptor changes. Specifically, in the presence of physiological E2 and therefore ERβ activation, serotonin preferentially acts on 5HT_2A receptors and to reduce vasospasm in cardiac tissue. After menopause, when 5HT_2A receptors have been down regulated, serotonin instead acts on 5HT_1A receptors, which cause adrenergic stimulation of smooth muscle92 and increase likelihood of cardiac vasospasm 93. This increases the risk of heart attack 92949596. In addition, testosterone, which increases following menopause, compounds the actions of serotonin at 5HT_1A receptors by preventing desensitization of 5HT_1A receptors 97. These changes in sensitivity of cardiac vessels, combined with increased clotting and lipid levels, would be expected to increase heart attack risk, arteriosclerosis and strokes. However, E2 is not solely responsible for protection from heart attack, progesterone also plays a role. Hormone replacement therapy (HRT) containing E2 and medroxyprogesterone instead of E2 and progesterone has been shown to increase heart attack 98. Although the study showing increased heart attack risk during HRT is controversial,99 it is possible that decreased concentrations of serotonin produced by treatment with medroxyprogesterone93100 could contribute to this increased risk.

Both E2 and serotonin are also active in the immune system, and in this system, their interaction is well-documented. E2 suppresses major histocompatibility complex II (MHC II) proteins in a tissue-specific manner 101 and acts centrally to suppress the immune system102 by helping to activate 5HT_2A receptors in the thymus 28103104105. Estrogen treatment also indirectly suppresses MHC II protein expression via serotonin 102106. Specifically, increased 5HT_2A activity causes decreased MHC II production,107 and decreased selection against self-reactive helper T cells (T_H1) 108. In addition, the concurrent inactivation of 5HT_1A receptors decreases TNF-α production 109110. Although self-reactive T_H1 cells are present, we hypothesize that E2's suppression of MHC II prevents them from becoming activated, and therefore while sufficient E2 is present they fail to attack tissues. Following menopause, or when E2 levels are unusually low, suppression of MHC II and immune function is lost, allowing self-reactive T_H1 cells to become active and pathogenic. It is possible that estrogen and serotonin's modulation of the immune system prevents immune attack on offspring during pregnancy (when estrogen is at relatively high concentrations) and avoids infection after delivery (when estrogen is relatively low) 111.

MHC II protein and self-reactive T cells appear to be the common denominators among autoimmune disorders in women, suggesting a role for E2 and serotonin in mediating these disorders. Multiple sclerosis (MS) is associated with the presence of MHC II protein polymorphic pathogenic alleles112113 and serotonin depletion114 This serotonin depletion could be a consequence of low E2, so the decrease in MS symptoms during pregnancy 115 could be explained by higher concentrations of E2. Also the severity of MS symptoms increases as serotonin levels decrease116, symptoms worsen in phases of the menstrual cycle when there is low E2117, and low levels of E2 result in changes in the 5HT signaling pathway 118. In female SERT knockout mice, symptoms of experimental allergic encephalomyelitis (a MS model) are less severe and have a greater latency to occurrence, possibly as a result of increased serotonin availability 119. Not only may low serotonin levels be linked to MS, but the effects of serotonin on MS may involve 5HT₂ receptors in particular. Gene-microarray analysis of brain lesions found lower 5HT₂ receptor expression in all 4 MS patients that analysis was preformed for compared to that of 2 controls 120.

Serotonin depletion could also be produced by conversion of serotonin to melatonin in the absence of light, which might explain the increased incidence of MS in more northern climates121 (where daylight periods are shorter) and the reason that light therapy can be effective in reducing symptoms of MS 122. Similarly, self-reported incidence of Type I diabetes (IDDM) is negatively correlated with exposure to UV radiation and positively correlated with latitude in Australia 123. Melatonin suppresses estrogen function 61 and suppresses 5HT_2A receptor activity 124. Further, melatonin might be the link between E2 and helper T-cell (T_H1) activity, as melatonin has been shown to upregulate expression of T_H1-stimulating factors such as TNF-α and IFN-γ 125. TNF-α increases the expression of MHC class II proteins and activates T_H1 cells, 126 which are hallmarks of MS.

Similar MHC class II polymorphisms and T cell dysfunctions have been implicated in lupus,127128 and lower levels of free tryptophan129 and MHC II protein over expression is also linked to autoimmune attack on beta cells in Type I diabetes (IDDM) 130. Over expression of the MHC II following failure to select against self-reactive T-cells is also a useful model for rheumatoid arthritis, Graves disease, and Hashimoto's thyroiditis, in which T-cells react to proteins produced in the body, failing to discriminate them from invading organisms 131. Women in whom estrogen-regulated serotonin signaling is compromised would be expected to have higher levels of MHC class II protein expression and may present these pathologies. However, simply over-expressing MHC II proteins is not sufficient to activate the immune system and induce autoimmune disorders 131. The links between autoimmune disorders, serotonergic systems, and E2 suggest that manipulation of serotonin or E2 could be used to successfully treat these pathologies. Consistent with this suggestion, ER agonists reduce the symptoms of autoimmune disorders 132133.

Carcinogenesis is conceptualized as consisting of three distinct phases: initiation, promotion and progression. Initiation is the irreversible alteration of a normal cell; promotion involves both proliferation of initiated cells and suppression of apoptosis of these cells; and progression is the irreversible conversion of one of the promoted initiated cells to an invasive, metastatic tumor cell 134. Therefore, any endogenous milieu that induces apoptosis or suppresses mitogenesis of initiated cells could reduce breast cancer risk.

For breast cancer, one of the prevailing theories for the role of E2 is that longer duration of lifetime exposure to E2 is associated with increased risk, so that early menarche and late menopause result in greater likelihood of developing breast cancer 135. Adding a role for serotonin does not conflict with this idea, but it does help explain several epidemiological findings that are not accounted for by a relationship between increased E2 exposure alone and breast cancer. First, the highest breast cancer incidence is in post-menopausal women, when endogenous E2 levels are much lower than before menopause. As described above, the higher E2 concentrations in the presence of progesterone prior to menopause cause an increase in 5HT_2A receptor density and serotonin activity that promotes apoptosis. In contrast, 5HT_1A activation (which occurs preferentially after menopause) decreases apoptotic signaling via caspase-3 suppression 38. Therefore, if E2 is acting on breast cancer in part by serotonin modulation, then we would predict that the decrease in E2 after menopause should increase risk of breast cancer. This is consistent with the observed breast cancer incidence curve 136. The failure of low levels of E2 to inhibit cancer growth is also reflected in patterns of tumor development within the estrous cycle. In mice, breast tumor growth occurs primarily in diestrus (when E2 is low), and tumor size is maintained or shrinks when E2 levels are high 137.

Second, in Pike's Breast Tissue Age model, a one-time rapid increase in breast tissue age and therefore breast cancer risk is included immediately following the first full-term pregnancy 138. The extension of Pike's model includes multiple births by incorporating smaller increases in risk at each additional full-term pregnancy 139. This pattern of increased risk for breast cancer immediately following full-term pregnancies is well-documented 140141142. E2 concentrations increase steadily during pregnancy, peaking at about 100 times normal cycling levels 3. In the days around parturition, these concentrations drop precipitously to levels below those of normal cycling females, where they are maintained for at least a month and potentially much longer (depending on suckling suppression) 143. We postulate that the observed increase in breast cancer risk may be accounted for by the concurrent decrease in E2 and therefore changes in 5HT_2A receptor function immediately prior to parturition. While E2's effect on serotonin could account for the immediate increase in risk, it cannot explain the long-term reduction in risk, which is likely related to other changes associated with parturition or lactation.

Third, obesity exerts differential effects on breast cancer risk over the lifespan; decreasing risk prior to menopause and increasing risk following menopause 144145. Under the prevailing theory of cumulative E2 exposure, obesity (which increases E2 levels146) would always be expected to increase breast cancer risk. However, the effect of E2 using serotonin mediation described above can account for the observed differential effects. Increased E2 in the presence of progesterone increases activation of 5HT_2A receptors, while increased E2 in the absence of progesterone increases activation of 5HT_1A receptors. The effects of these two receptors on apoptotic activity would predict that obesity exerts a protective effect before menopause and increases risk after menopause.

The importance of the presence of progesterone for this protective effect is underscored by recent HRT studies, which show that the use of estrogen and progesterone does not increase breast cancer risk,147 while the use of estrogen and medroxyprogesterone (which decreases serotonin in some tissues14148) has been shown to increase breast cancer risk. Consistent with the observed effects of HRT, oral contraception with Depo-Provera,^® which includes medroxyprogesterone rather than progesterone, has been shown to increase breast cancer risk [147, 149].