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This chapter reviews the neurobiological effects of stress sensitivity and s-citalpram (CIT) treatment observed in our nonhuman primate model of functional hypothalamic amenorrhea (FHA). This type of infertility, also known as stress-induced amenorrhea, is exhibited by cynomolgus macaques. In small populations, some individuals are stress-sensitive (SS) and others are highly stress-resilient (HSR). The SS macaques have suboptimal secretion of estrogen and progesterone during normal menstrual cycles. SS monkeys also have decreased serotonin gene expression and increased CRF expression compared to HSR monkeys. Recently, we found that CIT treatment improved ovarian steroid secretion in SS monkeys, but had no effect in HSR monkeys. Examination of the serotonin system revealed that SS monkeys had significantly lower Fev (fifth Ewing variant, rodent Pet1), TPH2 (tryptophan hydroxylase 2), 5HT1A autoreceptor and SERT (serotonin reuptake transporter) expression in the dorsal raphe than SR monkeys. However, CIT did not alter the expression of either Fev, TPH2, SERT or 5HT1A mRNAs. In contrast, SS monkeys tended to have a higher density of CRF fiber innervation of the dorsal raphe than HSR monkeys, and CIT significantly decreased the CRF fiber density in SS animals. In addition, CIT increased CRF-R2 gene expression in the dorsal raphe. We speculate that in a 15-week time frame, the therapeutic effect of S-citalopram may be achieved through a mechanism involving extracellular serotonin inhibition of CRF and stimulation of CRF-R2, rather than alteration of serotonin-related gene expression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: To use a nonhuman primate model and determine whether individuals sensitive to stress-induced reproductive dysfunction have lower activity of central serotonergic neurons under nonstressed conditions., Design: The activity of the central serotonergic system was assessed by measuring responsiveness to a fenfluramine challenge (5 mg/kg, IV) in sedated monkeys previously categorized as highly stress resistant (HSR; n = 4; normal menstrual cyclicity through two stress cycles), medium stress resistant (MSR; n = 5; ovulatory in the first stress cycle but anovulatory in the second stress cycle), or low stress resistant (i.e., stress sensitive, SS; n = 4; anovulatory as soon as stress is initiated). To control for differences in pituitary stores of prolactin or ACTH, the animals were subsequently administered a bolus of thyrotropin-releasing hormone (3 microg/kg) plus corticotropin releasing factor (CRF), (3 microg/kg)., Setting: Oregon National Primate Research Center, Animal Services Building., Patient(s): Female cynomolgus macaques exhibiting normal menstrual cycles., Intervention(s): Administration of fenfluramine, a serotonin-releasing drug., Main Outcomes Measure(s): Serum concentrations of prolactin (PRL) and cortisol (F)., Result(s): Prolactin release in response to fenfluramine was significantly greater in the HSR group compared with the MSR or SS groups. In contrast, cortisol was higher in the SS group compared with the other two groups. Similar responses were not evident after thyrotropin-releasing hormone + CRF stimulation., Conclusion(s): The lower PRL response to fenfluramine in the stress-sensitive animals suggests that stress-sensitive individuals have decreased activity in central serotonergic neurons. However, the F data suggest that the hypothalamic-pituitary-adrenal axis in stress-sensitive individuals is highly responsive to even small increases in serotonin.

Background/Aims: We established a cynomolgus macaque model of stress-induced amenorrhea in which the application of combined metabolic and psychosocial stress suppressed ovulation in stress-sensitive (SS) individuals, but not in highly stress-resilient (HSR) individuals. We previously reported that SS monkeys have deficits in global serotonin release and serotonin-related gene expression in the raphe nucleus, and that administration of the selective serotonin reuptake inhibitor S-citalopram increased estrogen and progesterone production in SS monkeys. In this study, we questioned whether there was a difference in corticotropin-releasing factor (CRF) or urocortin (UCN) stress-related peptide systems in the midbrain raphe region when HSR and SS monkeys treated with placebo or S-citalopram are compared. Methods: Monkeys characterized as HSR or SS were administered placebo or S-citalopram for 15 weeks. CRF fibers in the dorsal raphe were detected with an antibody against human CRF. UCN1 fibers were immunostained in an area rostral to the dorsal raphe. The fibers were quantified by stereology and analyzed by two-way ANOVA. UCN1 cell bodies were counted in the supraoculomotor area near the Edinger-Westphal nucleus. Results: S-citalopram significantly decreased the CRF fiber density in SS animals, but not in HSR animals. SS monkeys had a significantly lower UCN1 fiber density compared to HSR monkeys, but S-citalopram treatment did not alter the UCN1 fiber density. SS animals treated with S-citalopram tended to have a higher number of UCN1-positive cell bodies than the other groups. Conclusion: S-citalopram decreased CRF fiber density and appears to increase the production of UCN1 in SS individuals, indicating the likelihood that serotonin is involved in regulating CRF and UCN1 in individuals who are sensitive to the effects of serotonin. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: A body of knowledge implicates an increase in output from the locus ceruleus (LC) during stress. We questioned the innervation and function of the LC in our macaque model of Functional Hypothalamic Amenorrhea, also known as Stress-Induced Amenorrhea. Cohorts of macaques were initially characterized as highly stress resilient (HSR) or stress-sensitive (SS) based upon the presence or absence of ovulation during a protocol involving 2 menstrual cycles with psychosocial and metabolic stress. Afterwards, the animals were rested until normal menstrual cycles resumed and then euthanized on day 5 of a new menstrual cycle [a] in the absence of further stress; or [b] after 5days of resumed psychosocial and metabolic stress. In this study, parameters of the LC were examined in HSR and SS animals in the presence and absence of stress (2×2 block design) using ICC and image analysis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of catecholamines; and the TH level was used to assess by inference, NE output. The pixel area of TH-positive dendrites extending outside the medial border of the LC was significantly increased by stress to a similar degree in both HSR and SS animals (p<0.0001). There is a significant CRF innervation of the LC. The positive pixel area of CRF boutons, lateral to the LC, was higher in SS than HSR animals in the absence of stress. Five days of moderate stress significantly increased the CRF-positive bouton pixel area in the HSR group (p<0.02), but not in the SS group. There is also a significant serotonin innervation of the LC. A marked increase in medial serotonin dendrite swelling and beading was observed in the SS+stress group, which may be a consequence of excitotoxicity. The dendrite beading interfered with analysis of axonal boutons. However, at one anatomical level, the serotonin-positive bouton area was obtained between the LC and the superior cerebellar peduncle. Serotonin-positive bouton pixel area was significantly higher in HSR than SS animals (p<0.04). There was no change in either group after 5days of moderate stress. The ratio of serotonin/TH correlates with ovarian estrogen production with a sensitivity×stress interaction. Therefore, it appears that the serotonin system determines stress sensitivity and the NE system responds to stress. We hypothesize that elevated NE with low serotonin functionality ultimately leads to stress-induced infertility. In contrast, high serotonin functionality maintains ovulation in the presence of stress even with elevated NE. [Copyright &y& Elsevier]

Abstract: Like women, female cynomolgus monkeys show differential sensitivity to stress-induced reproductive dysfunction. A combined social and metabolic stress (mild diet+moderate exercise+relocation) will rapidly induce anovulation in a third of female cynomolgus monkeys (stress-sensitive; SS); a third will ovulate once and then become anovulatory (medium stress-resilient; MSR) and a third are highly stress-resilient (HSR) and exhibit normal menstrual cycles through two stressed menstrual cycles. In a non-stressed menstrual cycle, SS animals have lower levels of estrogen and progesterone, lower activity of the serotonin system and lower expression of genes related to the serotonin system in the dorsal raphe nucleus. In this study, we examined the expression of 5HT1A, 5HT2A, 5HT2C receptors and GAD67 in the hypothalamus of SS, HSR and MSR monkeys using in situ hybridization. SS monkeys exhibited higher expression of 5HT2A mRNA in the paraventricular nucleus (PVN), higher expression of 5HT2C and GAD67 in the infundibulum, as well as higher expression of GAD67 in the posterior hypothalamus (PH), compared with HSR monkeys. However, the expression of 5HT1A mRNA in the ventromedial nucleus (VMN) was not different between groups. We speculate that the serotonin and GABA systems may be altered in the stress–response and reproductive-related circuits of SS monkeys, and may be participating in altering the sensitivity of the reproductive system to stress in these individuals. [Copyright &y& Elsevier]

Abstract: Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. When stressed with a paradigm of relocation and diet for 60days, or 2 menstrual cycles, highly stress resilient monkeys continue to ovulate during both stress cycles (HSR); medium stress resilient monkeys ovulate once (MSR) and stress sensitive monkeys do not ovulate for the entire 60days (SS). This study examines serotonin-related gene expression in monkeys with different sensitivity to stress and exposed to 5days of moderate stress. Monkeys were first characterized as HSR, MSR or SS. After resumption of menstrual cycles, each monkey was re-stressed for 5days in the early follicular phase. The expression of 3 genes pivotal to serotonin neural function was assessed in the 3 groups of monkeys (n=4–5/group). Tryptophan hydroxylase 2 (TPH2), the serotonin reuptake transporter (SERT), and the 5HT1A autoreceptor mRNAs expression were determined at 4 morphological levels of the dorsal raphe nucleus with in situ hybridization (ISH) using digoxigenin-incorporated riboprobes. In addition, cFos was examined with immunohistochemistry. Positive pixel area and/or cell number were measured. All data were analyzed with ANOVA (3 groups) and with a t-test (2 groups). After 5days of stress, TPH2, SERT, 5HT1A and cFos were significantly lower in the SS group than the HSR group (p<0.05, all). This pattern of expression was the same as the pattern observed in the absence of stress in previous studies. Therefore, the ratio of the HSR/SS expression of each serotonergic gene was calculated in the presence and absence of stress. There was little or no difference in the ratio of HSR/SS gene expression in the presence or absence of stress. Moreover, cFos expression indicates that overall, cell activation in the dorsal raphe nucleus and periaquaductal gray is lower in SS than HSR animals. These data suggest that the serotonin system may set the sensitivity or resilience of the individual, but serotonin-related gene expression may not rapidly respond to moderate stress in nonhuman primates. [Copyright &y& Elsevier]

Objective: To study the effect of moderate stress on corticotropin-releasing factor (CRF) components in the serotonergic midbrain region in a monkey model of functional hypothalamic amenorrhea. Design: After characterization of stress sensitivity, monkeys were moved to a novel room and given 20% less chow for 5 days before euthanasia. Setting: Primate research center. Animal(s): Female cynomolgus macaques (Macaca fascicularis) characterized as highly stress resilient (HSR, n = 5), medium stress resilient (n = 4), or stress sensitive (SS, n = 4). Intervention(s): Five days of diet in a novel room with unfamiliar conspecifics. Main Outcome Measure(s): Density of CRF axons in the serotonergic dorsal raphe nucleus; the number of urocortin 1 (UCN1) cells; the density of UCN1 axons; the expression of CRF receptor 1 (CRF-R1) and CRF-R2 in the dorsal raphe nucleus. Result(s): The CRF innervation was higher in HSR than in SS animals; UCN1 cell number was higher in HSR than in SS animals and UCN1 axon bouton density was not different; all opposite of nonstressed animals. The CRF-R1 was not different between the sensitivity groups, but CRF-R2 was higher in HSR than in SS animals. The relative expression of CRF-R1 and CRF-R2 was similar to nonstressed animals. Conclusion(s): The HSR animals respond to stress with an increase in CRF delivery to serotonin neurons. With stress, UCN1 transport decreases in HSR animals. The CRF receptor expression was similar with or without stress. These changes may contribute to resilience in HSR animals. [ABSTRACT FROM AUTHOR]