Androgen metabolites impact CSF amines and axonal serotonin via MAO-A and -B in male macaques.

A number of studies have shown that mutations or deletions of the monoamine oxidase-A (MAO-A) gene cause elevated CNS serotonin and elevated impulsive aggression in humans and animal models. In addition, low cerebrospinal fluid (CSF) 5-hydroxyindole acetic acid (5HIAA) has been documented in a limited number of violent criminal populations and in macaques that exhibit impulsive aggression. To reconcile these different analyses, we hypothesized that CSF 5HIAA reflected degradation of serotonin by the activity of MAO-A; and that low MAO-A activity would result in lower CSF 5HIAA, but overall higher serotonin in the CNS. To test this hypothesis, male Japanese macaques (Macaca fuscata) were castrated, rested for 5-7months, and then treated for 3months with [1] placebo, [2] testosterone (T), [3] dihydrotestosterone (DHT; non-aromatizable androgen) and 1,4,6-androstatriene-3,17-dione (ATD) (steroidal aromatase inhibitor), or [4] flutamide (FLUT; androgen antagonist) and ATD (n=5/group). These treatments enable isolation of androgen and estrogen activities. In the dorsal raphe, MAO-A and MAO-B expressions were determined with in situ hybridization (ISH) and protein expression of aromatase was determined with immunohistochemistry (IHC). CSF concentrations of 5HIAA, 3-methoxy-4-hydroxyphenylglycol (MHPG), and homovanillic acid (HVA) were determined with liquid chromatography/mass spectrometry (LC/MS). From the same animals, previously published data on serotonin axon density were used as a proxy for CNS serotonin. Aromatase conversion of T to estrogen (E) suppressed MAO-A (positive pixel area, p=0.0045), but androgens increased MAO-B (positive pixel area, p=0.014). CSF 5HIAA was suppressed by conversion of T to E (Cohen's d=0.6). CSF 5HIAA was positively correlated with MAO-A-positive pixel area (r(2)=0.78). CSF 5HIAA was inversely correlated with serotonin axon-positive pixel area (r(2)=0.69). In summary, CSF 5HIAA reflects MAO-A activity rather than global serotonin. Low CSF 5HIAA may, in this paradigm, reflect higher serotonin activity. Androgens lower MAO-A activity via metabolism to E, thus elevating CNS serotonin and decreasing CSF 5HIAA. Since androgens increase certain types of aggression, these data are consistent with studies demonstrating that lower MAO-A activity is associated with elevated serotonin and increased aggression.

Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques.

Depression often accompanies the perimenopausal transition and it often precedes overt symptomology in common neurodegenerative diseases (NDDs, such as Alzheimer's, Parkinson's, Huntington, amyotrophic lateral sclerosis). Serotonin dysfunction is frequently found in the different etiologies of depression. We have shown that ovariectomized (Ovx) monkeys treated with estradiol (E) for 28 days supplemented with placebo or progesterone (P) on days 14-28 had reduced DNA fragmentation in serotonin neurons of the dorsal raphe nucleus, and long-term Ovx monkeys had fewer serotonin neurons than intact controls. We questioned the effect of E alone or E+P (estradiol supplemented with progesterone) on gene expression related to DNA repair, protein folding (chaperones), the ubiquitin-proteosome, axon transport and NDD-specific genes in serotonin neurons. Ovx macaques were treated with placebo, E or E+P (n=3 per group) for 1 month. Serotonin neurons were laser captured and subjected to microarray analysis and quantitative real-time PCR (qRT-PCR). Increases were confirmed with qRT-PCR in five genes that code for proteins involved in repair of strand breaks and nucleotide excision. NBN1, PCNA (proliferating nuclear antigen), GADD45A (DNA damage-inducible), RAD23A (DNA damage recognition) and GTF2H5 (gene transcription factor 2H5) significantly increased with E or E+P treatment (all analysis of variance (ANOVA), P<0.01). Chaperone genes HSP70 (heat-shock protein 70), HSP60 and HSP27 significantly increased with E or E+P treatment (all ANOVA, P<0.05). HSP90 showed a similar trend. Ubiquinase coding genes UBEA5, UBE2D3 and UBE3A (Parkin) increased with E or E+P (all ANOVA, P<0.003). Transport-related genes coding kinesin, dynein and dynactin increased with E or E+P treatment (all ANOVA, P<0.03). SCNA (α-synuclein) and ADAM10 (α-secretase) increased (both ANOVA, P<0.02) but PSEN1 (presenilin1) decreased (ANOVA, P<0.02) with treatment. APP decreased 10-fold with E or E+P administration. Newman-Keuls post hoc comparisons indicated variation in the response to E alone versus E+P across the different genes. In summary, E or E+P increased gene expression for DNA repair mechanisms in serotonin neurons, thereby rendering them less vulnerable to stress-induced DNA fragmentation. In addition, E or E+P regulated four genes encoding proteins that are often misfolded or malfunctioning in neuronal populations subserving overt NDD symptomology. The expression and regulation of these genes in serotonergic neurons invites speculation that they may mediate an underlying disease process in NDDs, which in turn may be ameliorated or delayed with timely hormone therapy in women.

Relationships between androgens, serotonin gene expression and innervation in male macaques.

Androgen administration to castrated individuals was purported to decrease activity in the serotonin system. However, we found that androgen administration to castrated male macaques increased fenfluramine-induced serotonin release as reflected by increased prolactin secretion. In this study, we sought to define the effects of androgens and aromatase inhibition on serotonin-related gene expression in the dorsal raphe, as well as serotonergic innervation of the LC. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with (1) placebo, (2) testosterone (T), (3) dihydrotestosterone (DHT; non-aromatizable androgen) and ATD (steroidal aromatase inhibitor), or (4) Flutamide (FLUT; androgen antagonist) and ATD (n=5/group). This study reports the expression of serotonin-related genes: tryptophan hydroxylase 2 (TPH2), serotonin reuptake transporter (SERT) and the serotonin 1A autoreceptor (5HT1A) using digoxigenin-ISH and image analysis. To examine the production of serotonin and the serotonergic innervation of a target area underlying arousal and vigilance, we measured the serotonin axon density entering the LC with ICC and image analysis. TPH2 and SERT expression were significantly elevated in T- and DHT + ATD-treated groups over placebo- and FLUT + ATD-treated groups in the dorsal raphe (p < 0.007). There was no difference in 5HT1A expression between the groups. There was a significant decrease in the pixel area of serotonin axons and in the number of varicosities in the LC across the treatment groups with T > placebo > DHT + ATD = FLUT + ATD treatments. Comparatively, T- and DHT + ATD-treated groups had elevated TPH2 and SERT gene expression, but the DHT + ATD group had markedly suppressed serotonin axon density relative to the T-treated group. Further comparison with previously published data indicated that TPH2 and SERT expression reflected yawning and basal prolactin secretion. The serotonin axon density in the LC agreed with the area under the fenfluramine-stimulated prolactin curve, providing a morphological basis for the pharmacological results. This suggested that androgen activity increased TPH2 and SERT gene expression but, aromatase activity, and neural production of estradiol (E), may subserve axonal serotonin and determination of the compartment acted upon by fenfluramine. In summary, androgens stimulated serotonin-related gene expression, but aromatase inhibition dissociated gene expression from the serotonin innervation of the LC terminal field and fenfluramine-stimulated prolactin secretion.

Hypothalamic KISS1 expression, gonadotrophin-releasing hormone and neurotransmitter innervation vary with stress and sensitivity in macaques.

The present study examined the effect of short-term psychosocial and metabolic stress in a monkey model of stress-induced amenorrhaea on the hypothalamic-pituitary-gonadal axis. KISS1 expression was determined by in situ hybridisation in the infundibular arcuate nucleus. Downstream of KISS1, gonadotrophin-releasing hormone (GnRH) axons in lateral areas rostral to the infundibular recess, serum luteinising hormone (LH) and serum oestradiol were measured by immunohistochemistry and radioimmunoassay. Upstream of KISS1, norepinephrine axons in the rostral arcuate nucleus and serotonin axons in the anterior hypothalamus and periaqueductal grey were measured by immunohistochemistry. Female cynomolgus macaques (Macaca fascicularis) characterised as highly stress resilient (HSR) or stress sensitive (SS) were examined. After characterisation of stress sensitivity, monkeys were either not stressed, or mildly stressed for 5 days before euthanasia in the early follicular phase. Stress consisted of 5 days of 20% food reduction in a novel room with unfamiliar conspecifics. There was a significant increase in KISS1 expression in HSR and SS animals in the presence versus absence of stress (P = 0.005). GnRH axon density increased with stress in HSR and SS animals (P = 0.015), whereas LH showed a gradual but nonsignificant increase with stress. Oestradiol trended higher in HSR animals and there was no effect of stress (P = 0.83). Norepinephrine axon density (marked with dopamine ß-hydroxylase) increased with stress in both HSR and SS groups (P ≤ 0.002), whereas serotonin axon density was higher in HSR compared to SS animals and there was no effect of stress (P = 0.03). The ratio of dopamine ß-hydroxylase/oestradiol correlated with KISS1 (P = 0.052) and GnRH correlated with serum LH (P = 0.039). In conclusion, oestradiol inhibited KISS1 in the absence of stress, although stress increased norepinephrine, which may over-ride oestradiol inhibition of KISS1 expression. We speculate that neural pathways transduce stress to KISS1 neurones, which changes their sensitivity to oestradiol.

Ovarian regulation of kisspeptin neurones in the arcuate nucleus of the rhesus monkey (macaca mulatta).

Tonic gonadotrophin secretion throughout the menstrual cycle is regulated by the negative-feedback actions of ovarian oestradiol (E2) and progesterone. Although kisspeptin neurones in the arcuate nucleus (ARC) of the hypothalamus appear to play a major role in mediating these feedback actions of the steroids in nonprimate species, this issue has been less well studied in the monkey. In the present study, we used immunohistochemistry and in situ hybridisation to examine kisspeptin and KISS1 expression, respectively, in the mediobasal hypothalamus (MBH) of adult ovariectomised (OVX) rhesus monkeys. We also examined kisspeptin expression in the MBH of ovarian intact females, and the effect of E2, progesterone and E2 + progesterone replacement on KISS1 expression in OVX animals. Kisspeptin or KISS1 expressing neurones and pronounced kisspeptin fibres were readily identified throughout the ARC of ovariectomised monkeys but, on the other hand, in intact animals, kisspeptin cell bodies were small in size and number and only fine fibres were observed. Replacement of OVX monkeys with physiological levels of E2, either alone or with luteal phase levels of progesterone, abolished KISS1 expression in the ARC. Interestingly, progesterone replacement alone for 14 days also resulted in a significant down-regulation of KISS1 expression. These findings support the view that, in primates, as in rodents and sheep, kisspeptin signalling in ARC neurones appears to play an important role in mediating the negative-feedback action of E2 on gonadotrophin secretion, and also indicate the need to study further their regulation by progesterone.

Ovarian steroids increase spinogenetic proteins in the macaque dorsal raphe.

Dendritic spines are the basic structural units of neuronal plasticity. Intracellular signaling cascades that promote spinogenesis have centered on RhoGTPases. We found that ovarian steroids increase gene expression of RhoGTPases [Ras homolog gene family member A (RhoA), cell division control protein 42 homolog (Cdc42), and ras-related C3 botulinum toxin substrate (Rac)] in laser-captured serotonin neurons. We sought to confirm that the increases observed in gene expression translate to the protein level. In addition, a preliminary study was conducted to determine whether an increase in spines occurs via detection of the spine marker protein, postsynaptic density-95 (PSD-95). Adult ovariectomized (Ovx) monkeys were treated with estradiol (E), progesterone (P), or E+P for 1 month. Sections through the dorsal raphe nucleus were immunostained for RhoA and Cdc42 (n=3-4/group). The number and positive pixel area of RhoA-positive cells and the positive pixel area of Cdc42-positive fibers were determined. On combining E- and E+P-treated groups, there was a significant increase in the average and total cell number and positive pixel area of RhoA-positive cells. E, P, and E+P treatments, individually or combined, also increased the average and total positive pixel area of Cdc42-positive fibers. With remaining sections from two animals in each group, we conducted a preliminary examination of the regulation of PSD-95 protein expression. PSD-95, a postsynaptic scaffold protein, was examined with immunogold silver staining (n=2/group), and the total number of PSD-95-positive puncta was determined with stereology across four levels of the dorsal raphe. E, P, and E+P treatment significantly increased the total number of PSD-95-positive puncta. Together, these findings indicate that ovarian steroids act to increase gene and protein expression of two pivotal RhoGTPases involved in spinogenesis and preliminarily indicate that an increased number of spines and/or synapses result from this action. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and in turn, increase serotonin neuronal activity throughout the brain.

5-HTTLPR genotype and gender, but not chronic fluoxetine administration, are associated with cortical TREK1 protein expression in rhesus macaques.

TREK1 is a widely expressed background potassium channel. Similar to mice treated with selective serotonin reuptake inhibitors (SSRIs), TREK1 knockout mice are resistant to depression-like behavior and have elevated serotonin levels leading to speculation that TREK1 inhibition may contribute to the therapeutic effects of SSRIs. This study examined how chronic fluoxetine administration and a common functional polymorphism in the serotonin-transporter-linked promoter region (5-HTTLPR) influence cortical TREK1 expression in 24 rhesus monkeys. The short rh5-HTTLPR allele as well as female gender were associated with reduced cortical TREK1 protein expression but chronic SSRI administration had no effect. These results suggest that serotonin may influence TREK1, but that chronic SSRI treatment does not result in long lasting changes in cortical TREK1 protein expression. TREK1 gender differences may be related to gender differences in serotonin and require further research.

Long-term ovariectomy decreases serotonin neuron number and gene expression in free ranging macaques.

The serotonin system responds to the ovarian steroids, estradiol (E) and progesterone (P), in women and female animal models. In macaques, ovarian steroid administration to ovariectomized (Ovx) individuals improves serotonin neural function through actions on pivotal serotonin-related genes and proteins, such as TPH2 (tryptophan hydroxylase 2), SERT (serotonin reuptake transporter), and the 5HT1A autoreceptor. In addition, ovarian steroid administration reduces gene and protein expression in the caspase-independent pathway and reduces DNA fragmentation in serotonin neurons. This study examines the hypothesis that long-term ovariectomy will lead to a loss of serotonin neurons and compromised gene expression in serotonin neurons. Female Japanese macaques were ovariectomized or tubal ligated (n=5/group) at 3 years of age and returned to their natal troop. After 3 years, the animals were collected, administered a fenfluramine challenge to determine global serotonin availability, and then euthanized. Fev, TPH2, SERT, and 5HT1A expression were examined with digoxigenin in situ hybridization (ISH) and quantitative image analysis. Cell number, positive pixel area, and average pixel density were determined. In the Ovx group, Fev, TPH2, SERT, and 5HT1A showed a significant decease in average and total cell number and positive pixel area. The reduction in Fev-positive neurons suggests that there were fewer serotonin neurons in Ovx animals compared to ovary-intact animals. The decrease in TPH2 in the Ovx animals was consistent with earlier results in 5-month Ovx animals, but it may be due to the decrease in cell number rather than a decrease in expression on an individual cell basis. The decrease in SERT and 5HT1A in long-term Ovx differed from previous studies in short-term Ovx. In summary, long-term ovarian steroid loss resulted in fewer serotonin neurons and overall lower Fev, TPH2, SERT, and 5HT1A gene expression. This may be due to serotonin cell death or to a negative impact on a long-term developmental process in young female macaques.

Ovarian steroid regulation of the midbrain corticotropin releasing factor and urocortin systems in macaques.

A significant number of postmenopausal women report increased anxiety and vulnerability to stress, which has been linked to decreased secretion of ovarian steroids. Communication between the serotonin system and the corticotropin releasing factor (CRF) system determines stress sensitivity or resilience. This study examines the effects of the ovarian steroids, estradiol (E) and progesterone (P) on the CRF system components that impact serotonin neurons in the midbrain of nonhuman primates. Ovariectomized rhesus macaques were treated with placebo, E alone for 1 month, or E supplemented with P for the last 2 weeks. Quantitative (q)RT-PCR and immunocytochemistry were employed. E±P treatment decreased CRF-R1 and increased CRF-R2 gene expression in hemi-midbrain blocks and in laser captured serotonin neurons. Also in hemi-midbrains, E treatment increased urocortin 1 (UCN1) and CRFBP gene expression, but supplemental P treatment reversed these effects. E±P decreased CRF fiber density in the dorsal, interfascicular and median raphe nuclei and decreased CRF-R1 immunostaining in the dorsal raphe. E increased CRF-R2 immunostaining in the dorsal and median raphe. E±P increased UCN1 immunostaining in the cell bodies and increased UCN1 fiber density in the caudal linear nucleus. Estrogen receptor beta (ERß), but not ERα was detected in the nucleus of UCN1-positive neurons. While the mechanism of ovarian hormone regulation of the midbrain CRF system requires further investigation, these studies clearly demonstrate another pathway by which ovarian hormones may have positive effects on anxiety and mood regulation.

Ovarian steroids decrease DNA fragmentation in the serotonin neurons of non-injured rhesus macaques.

We previously found that ovarian steroids promote neuroprotection in serotonin neurons by decreasing the expression of pro-apoptotic genes and proteins in the dorsal raphe nucleus of rhesus macaques, even in the absence of overt injury. In this study, we questioned whether these actions would lead to a reduction in DNA fragmentation in serotonin neurons. Ovariectomized (OVX) rhesus monkeys were implanted with silastic capsules that were empty (placebo) or containing estradiol (E), progesterone (P) or estradiol and progesterone (E+P) for 1 month. In all animals, eight levels of the dorsal raphe nucleus in a rostral-to-caudal direction were immunostained using the terminal deoxynucleotidyl transferase nick end labeling (TUNEL) method. Two staining patterns were observed, which are referred to as type I, with complete dark staining of the nucleus, and type II, with peripheral staining in the perinuclear area. A montage of the dorsal raphe was created at each level with a Marianas Stereology Microscope and Slidebook 4.2, and the TUNEL-positive cells were counted. In direct comparison with OVX animals, P treatment and E+P treatment significantly reduced the total number of TUNEL-positive cells (Mann-Whitney test, both treatments P=0.04) and E+P treatment reduced the number of TUNEL-positive cells per mm(3) (Mann-Whitney test, P=0.04). Double immunocytochemistry for TUNEL and tryptophan hydroxylase (TPH) indicated that DNA fragmentation was prominent in serotonin neurons. These data suggest that in the absence of ovarian steroids, a cascade of gene and protein expression leads to an increase in DNA fragmentation in serotonin neurons. Conversely, ovarian steroids have a neuroprotective role in the non-injured brain and prevent DNA fragmentation and cell death in serotonin neurons of nonhuman primates.

Effect of ovarian hormones on genes promoting dendritic spines in laser-captured serotonin neurons from macaques.

Dendritic spines are the elementary structural units of neuronal plasticity and the cascades that promote dendritic spine remodeling center on Rho GTPases and downstream effectors of actin dynamics. In a model of hormone replacement therapy, we sought the effect of estradiol (E) and progesterone (P) on gene expression in these cascades in laser-captured serotonin neurons from rhesus macaques with complementary DNA array analysis. Ovariectomized rhesus macaques were treated with either placebo, E or E+P through Silastic implant for 1 month before euthanasia. The midbrain was obtained, sectioned and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (PixCell II). RNA from laser-captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips. With E±P treatment, there was a significant change in 744 probe sets (analysis of variance, P<0.05), but 10,493 probe sets exhibited a twofold or greater change. Pivotal changes in pathways leading to dendritic spine proliferation and transformation included twofold or greater increases in expression of the Rho GTPases called CDC42, Rac1 and RhoA. In addition, twofold or greater increases occurred in downstream effectors of actin dynamics, including p21-activated kinase (PAK1), Rho-associated coiled-coil-containing protein kinase (ROCK), PIP5K, IRSp53, Wiskott-Aldrich syndrome protein (WASP), WASP family Verprolin-homologous protein (WAVE), MLC, cofilin, gelsolin, profilin and three subunits of actin-related protein (ARP2/3). Finally, twofold or greater decreases occurred in CRIPAK, LIMK2 and myosin light chain kinase (MLCK). The regulation of RhoA, Rac1, CDC42, ROCK, PIP5k, IRSp53, WASP, WAVE, LIMK2, CRIPAK1, MLCK, ARP2/3 subunit 3, gelsolin, profilin and cofilin was confirmed with nested quantitative reverse transcriptase-PCR on laser-captured RNA (n=3 animals/treatment). The data indicate that ovarian steroids target gene expression of the Rho GTPases and pivotal downstream proteins, that in turn would promote dendritic spine proliferation and stabilization on serotonin neurons of the dorsal raphe nucleus.

Stress sensitive female macaques have decreased fifth Ewing variant (Fev) and serotonin-related gene expression that is not reversed by citalopram.

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 60 days or two menstrual cycles, highly stress resilient monkeys (HSR) continued to ovulate during the stress cycles whereas stress sensitive monkeys (SS) did not. After cessation of stress, monkeys characterized as HSR or SS were administered placebo (PL) or S-citalopram (CIT) for 15 weeks at doses that normalized ovarian steroid secretion in the SS animals and that maintained blood CIT levels in a therapeutic range. After euthanasia, the brain was perfused with 4% paraformaldehyde. The pontine midbrain was blocked and sectioned at 25 microm. The expression of four genes pivotal to serotonin neural function was assessed in the four groups of monkeys (n=4/group). Fev (fifth Ewing variant) ETS transcription factor, tryptophan hydroxylase 2 (TPH2), the serotonin reuptake transporter (SERT), and the 5HT1A autoreceptor were determined at 7-8 levels of the dorsal raphe nucleus with in situ hybridization (ISH) using radiolabeled- and digoxygenin-incorporated riboprobes. Positive pixel area and cell number were measured with Slidebook 4.2 in the digoxigenin assay for Fev. Optical density (OD) and positive pixel area were measured with NIH Image software in the radiolabeled assays for TPH2, SERT and 5HT1A. All data were analyzed with two-way ANOVA. SS monkeys had significantly fewer Fev-positive cells and lower Fev-positive pixel area in the dorsal raphe than HSR monkeys. SS monkeys also had significantly lower levels of TPH2, SERT and 5HT1A mRNAs in the dorsal raphe nucleus than HSR monkeys. However, CIT did not alter the expression of either Fev, TPH2, SERT or 5HT1A mRNAs. These data suggest that SS monkeys have fewer serotonin (5-HT) neurons than HSR monkeys, and that they have deficient Fev expression, which in turn, leads to deficient TPH2, SERT and 5HT1A expression. In addition, the therapeutic effect of CIT is probably achieved through mechanisms other than alteration of 5-HT-related gene expression.

Differential effects of ovarian steroids and raloxifene on serotonin 1A and 2C receptor protein expression in macaques.

To further understand the role of ovarian hormones in the function of the serotonin neural system, we investigated the effects of estradiol (E), progesterone (P), and raloxifene on 5HT 1A and 2C receptor protein expression in the dorsal raphe region using Western blot analysis. Adult rhesus macaques (Macaca mulatta) were ovariectomized (Ovx) and implanted with Silastic capsules containing E or P. In the first paradigm, animals that had been Ovx for 6-16 months were treated for 1 month with E (El) or E + P (EP1) and compared to animals that were untreated and Ovx for 5 months (n = 4 per group). In the second paradigm, comparisons were made between animals that were Ovx and untreated for 5 months, or Ovx and immediately implanted with Silastic capsules containing E or E + P for 5 months (E5, EP5), or administered raloxifene in the diet for 5 months (Ral5) (n = 4 per group). The dorsal raphe region was harvested, homogenized and a crude membrane fraction was obtained for examination of receptor proteins. In the first paradigm, 5HT1A receptor protein expression was significantly lower in E1 and EPI treatment groups compared to the Ovx-control group (ANOVA P = 0.01; posthoc P < 0.03), but 5HT2C receptor expression was unaffected by 1 month of E or EP treatment. In the second paradigm, there was no difference in 5HT1A receptor expression between the Ovx-control group and the E5 group, but 5HT1A receptor expression was significantly suppressed in the EP5 group (ANOVA P = 0.04; posthoc P < 0.05). In addition, 5HT2C expression increased in the E5 treatment group relative to the Ovx-control group. Addition of P to the E5 regimen prevented the E5-induced increase in 5HT2C receptor expression and significantly reduced 5HT2C receptor expression to a level below that observed in the Ovx-control group (ANOVA P = 0.001; posthoc P < 0.05). Thus, 5HT1A receptor may lose sensitivity to the suppressive effect of E after 5 months, whereas the 5HT2C receptor increases. However, addition of P in the EP5 regimen maintains the regulatory effects observed with 1 month of treatment. 5HT1A receptor protein levels were higher with raloxifene treatment than in Ovx-control animals (P < 0.01), suggesting that raloxifene may antagonize residual E in Ovx animals.

Unique responses of midbrain CART neurons in macaques to ovarian steroids.

CART (cocaine and amphetamine regulated transcript) is a neuropeptide involved in the control of several physiological processes, such as response to psychostimulants, food intake, depressive diseases and neuroprotection. It is robustly expressed in the brain, mainly in regions that control emotional and stress responses and it is regulated by estrogen in the hypothalamus. There is a distinct population of CART neurons located in the vicinity of the Edinger-Westphal nucleus of the midbrain that also colocalize urocortin-1. The aims of this study were 1) to determine the distribution of CART immunoreactive neurons in the monkey midbrain, 2) to examine the effects of estrogen (E) and progesterone (P) on midbrain CART mRNA and peptide expression and 3) to determine whether midbrain CART neurons contain steroid receptors. Adult female rhesus monkeys (Macaca mulatta) were spayed and either treated with placebo (OVX), estrogen alone (E), progesterone alone (P) or E+P. Animals were prepared (a) for RNA extraction followed by microarray analysis and quantitative (q) RT-PCR (n=3/group); (b) for immunohistochemical analysis of CART and CART+tryptophan hydroxylase (TPH), CART+estrogen receptors (ER) or CART+progesterone receptors (n=5/group) and (c) for Western blots (n=3/group). Both E- and E+P-administration decreased CART gene expression on the microarray and with qRT-PCR. Stereological analysis of CART immunostaining at five levels of the Edinger-Westphal nucleus indicated little effect of E or E+P administration on the area of CART immunostaining. However, P administration increased CART-immunopositive area in comparison to the OVX control group with Student's t-test, but not with ANOVA. CART 55-102 detection on Western blot was unchanged by hormone administration. ERbeta and PR were detected in CART neurons and CART fibers appeared to innervate TPH-positive serotonin neurons in the dorsal raphe. In summary, E decreased CART mRNA, but this effect did not translate to the protein level. Moreover, P administration alone had a variable effect on CART mRNA, but it caused an increase in CART immunostaining. Together, the data suggest that CART neurons in the midbrain have a unique steroid response, which may be mediated by nuclear receptors, neuroactive steroids or interneurons.

Neuroprotective actions of ovarian hormones without insult in the raphe region of rhesus macaques.

Using a nonhuman primate model of surgical menopause, our laboratory has shown that ovarian hormone treatment (HT) improves 5-HT neural function in the dorsal raphe nucleus (DRN). We further hypothesize that HT may increase 5-HT neuronal resilience. Recent data from microarray analysis indicated that HT regulates gene expression in pathways that lead to apoptosis. In this study, we questioned whether HT alters protein expression in caspase-dependent and independent pathways. Ovariectomized monkeys received Silastic implants containing placebo (empty), estrogen (E) or E+ progesterone (P). A small block of the midbrain containing the DRN was dissected and subjected to subcellular fractionation, yielding cytosolic, nuclear and mitochondrial fractions (n=4/group). The pro-apoptotic protein, c-jun n-terminal kinase (JNK1) and its phosphorylation were decreased by E+P treatment in the cytosolic fraction. Downstream of JNK are proteins in the caspase-dependent and -independent pathways. First, in the caspase-dependent pathway, cytoplasmic and mitochondrial fractions were immunoblotted for Bcl-2 family members, cytochrome c, Apaf1 and XIAP. However, the expression of these proteins did not differ among treatments. Pro-caspase 3 was decreased by E+P, but there was no evidence of active caspase in any group. Then, we examined the involvement of a protein in the caspase-independent pathway, called apoptosis-inducing factor (AIF). AIF mRNA (n=3/group) and AIF mitochondrial protein tended to decrease with hormone treatment. However, AIF protein in the nuclear fraction in E+P treated monkeys was significantly reduced. This indicates that HT is reducing the translocation of AIF from mitochondria to nucleus, thus inhibiting AIF-mediated apoptosis. AIF was immunocytochemically localized to large 5-HT-like neurons of the dorsal raphe. These data suggest that in the absence of global trauma or ischemia, HT may act through the caspase-independent pathway to promote neuroprotection in the 5-HT system.

Hypothalamic gonadotrophin-releasing hormone expression in female monkeys with different sensitivity to stress.

Psychosocial stress, combined with mild dieting and moderate exercise, are observed in women seeking treatment for hypothalamic amenorrhea. Using female cynomolgus macaques, we previously reported that the same combination of mild stresses suppressed reproductive hormone secretion and menstrual cycles in some individuals (stress-sensitive, SS), but not in others (highly stress-resilient, HSR). Compared to HSR monkeys, SS monkeys exhibited lower oestradiol and progesterone levels at the midcycle peak and decreased gene expression in the central serotonergic system during nonstressed cycles. Because steroids and serotonin impinge upon the hypothalamic-pituitary-gonadal (HPG) axis, we hypothesised that the differences between SS and HSR monkeys in the sensitivity of the HPG axis to stress may ultimately manifest in differences in the gonadotrophin-releasing hormone (GnRH) system. GnRH in situ hybridisation and immunohistochemistry were performed with hypothalamic sections from SS and HSR animals, euthanised in the early follicular phase of a nonstressed menstrual cycle. Compared to HSR monkeys, SS monkeys exhibited a significantly higher number and density of GnRH cell bodies, as well as a higher number of soma with extremely robust expression of GnRH mRNA, but SS monkeys exhibited a lower density of immunostained GnRH fibres in the median eminence. We suggest that neuronal mechanisms involved in the control of GnRH synthesis, transport and release differ in SS compared to HSR animals.

Serotonin-related gene expression in female monkeys with individual sensitivity to stress.

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. In this study, the expression of four genes pivotal to serotonin neural function was assessed in monkeys previously categorized as highly stress resistant (n=3; normal menstrual cyclicity through two stress cycles), medium stress resistant (n=5; ovulatory in the first stress cycle but anovulatory in the second stress cycle), or low stress resistant (i.e. stress-sensitive; n=4; anovulatory as soon as stress is initiated). In situ hybridization and quantitative image analysis was used to measure mRNAs coding for SERT (serotonin transporter), 5HT1A autoreceptor, MAO-A and MAO-B (monoamine oxidases) at six levels of the dorsal raphe nucleus (DRN). Optical density (OD) and positive pixel area were measured with NIH Image software. In addition, serotonin neurons were immunostained and counted at three levels of the DRN. Finally, each animal was genotyped for the serotonin transporter long polymorphic region (5HTTLPR). Stress sensitive animals had lower expression of SERT mRNA in the caudal region of the DRN (P<0.04). SERT mRNA OD in the caudal DRN was positively correlated with serum progesterone during a pre-stress control cycle (P<0.0007). 5HT1A mRNA OD signal tended to decline in the stress-sensitive group, but statistical difference between averages was lacking in analysis of variance. However, 5HT1A mRNA signal was positively correlated with control cycle progesterone (P<0.009). There was significantly less MAO-A mRNA signal in the stress-sensitive group (P<0.007) and MAO-A OD was positively correlated with progesterone from a pre-stress control cycle (P<0.007). MAO-B mRNA exhibited a similar downward trend in the stress-sensitive group. MAO-B OD also correlated with control cycle progesterone (P<0.003). There were significantly fewer serotonin neurons in the stress-sensitive group. All animals contained only the long form of the 5HTTLPR. Thus, all serotonin-related mRNAs examined in the dorsal raphe to date were lower (SERT, MAO-A) or exhibited a lower trend (5HT1A, MAO-B) in the stress sensitive animals, which probably reflects the lower number of serotonin neurons present.

Ovarian hormone influences on the density of immunoreactivity for tyrosine hydroxylase and serotonin in the primate corpus striatum.

The serotonergic and dopaminergic inputs to the corpus striatum in human and non-human primates participate in diverse sensorimotor, cognitive, and affective functions, are implicated in dysfunction in diseases such as Parkinson's disease and schizophrenia, and are targets for many of the drugs used to treat these disorders. Sex differences in the incidence and/or clinical course of these disorders and in the effectiveness of related dopaminergic and serotonergic drug therapies suggest that primate striatal indolamines and catecholamines are also influenced by gonadal hormones. However, while well studied in rats, relatively little is known about precisely how gonadal steroids modulate stratial dopamine and serotonin systems in primates. To begin to address this issue, the present studies explored the effects of ovarian steroids on the serotonergic and dopaminergic innervation densities of the caudate, putamen, and the nucleus accumbens in young adult rhesus monkeys. Using densitometry to quantify immunoreactivity for serotonin and for the catecholamine-synthesizing enzyme tyrosine hydroxylase, innervation densities were compared in identified, functionally specialized striatal subdomains across animals that were either ovariectomized or ovariectomized and supplemented with estradiol and/or progesterone, i.e. in a primate model of surgical menopause, with and without hormone replacement therapy. These analyses revealed clear examples of structure-, hemisphere-, and replacement regimen-specific effects of changes in circulating steroids on the densities of each afferent system examined. Further, the predominantly stimulatory effects observed occurred in striatal areas analogous to those suspected as sites of localized dopamine and/or serotonin compromise in Parkinson's disease and schizophrenia. Thus, the hormone actions identified in this study could hold relevance for some of the sex differences identified in relation to these disorders, including the findings of decreased incidence and/or symptom severity in women that have led to hypotheses of protective effects for estrogen.

Soy and social stress affect serotonin neurotransmission in primates.

Stress and sex steroidal milieu can each influence mood in women. The purpose of this study was to compare the effect of long-term conjugated equine estrogen (CEE), soy phytoestrogen (SPE), and social subordination stress on dorsal raphe serotonin neurotransmission of ovariectomized cynomolgus monkeys. Tryptophan hydroxylase (TPH) and serotonin reuptake transporter (SERT) protein content were determined, and the in vitro degradation of macaque SERT protein was examined in the presence and absence of protease inhibitors, serotonin (5-HT), and citalopram. Like CEE, SPE increased TPH protein levels. Social subordinates had markedly lower TPH protein levels than dominants regardless of hormone replacement. Therefore, these two variables had independent and additive effects. CEE and SPE increased SERT, and social status had no effect. Thus, the hormone-induced increase in SERT was accompanied by increased 5-HT synthesis and neuronal firing, which appears biologically reasonable as 5-HT prevented SERT degradation in vitro.

Ovarian steroid regulation of serotonin reuptake transporter (SERT) binding, distribution, and function in female macaques.

The serotonin reuptake transporter (SERT) plays an important role in serotonin neurotransmission and in several psychopathological disorders such as depression and anxiety disorders. In this study, we investigated whether the ovarian steroids, estrogen (E) and progesterone (P) regulate SERT binding, intracellular distribution, and function using [(3)H]citalopram ligand binding with quantitative autoradiography, immunofluorescence histochemistry with confocal microscopy and [(3)H]serotonin uptake, respectively. Ovariectomized macaques received either placebo, E alone, P alone or E plus P for 28 days. In the raphe, E, P, and E+P treatments did not change SERT binding density. In several hypothalamic nuclei, [(3)H]citalopram binding was increased by E, P, and E+P. Immunofluorescent SERT in serotonin soma was intracellular and similar among treatments. In the hypothalamus, immunofluorescent SERT was located along the serotonergic axons and there was a significant proliferation of immunofluorescent fibers in hormone-treated animals. In addition, E and E+P treatment increased serotonin uptake in the basal ganglia. These findings suggest that ovarian hormones regulate SERT protein expression and distribution, perhaps via extracellular serotonin or mRNA stability, but not solely at the level of gene transcription. Further investigation on the possible action of ovarian steroids on the directionality of SERT transport is indicated.