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ABSTRACT: Aggression in humans and animals has been linked to androgens and serotonin function. To further our understanding of the effect of androgens on serotonin and aggression in male macaques, we sought to manipulate circulating androgens and the activity of aromatase; and to then determine behavior and the endogenous availability of serotonin. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with (a) placebo; (b) testosterone (T); (c) T + Dutasteride (5a reductase inhibitor; AvodartTM); (d) T + Letrozole (nonsteroidal aromatase inhibitor; FemeraTM); (e) Flutamide + ATD (androgen antagonist plus steroidal aromatase inhibitor); or (f) dihydrotestosterone (DHT) + ATD (n = 5/group). Behavioral observations were made during treatments. At the end of the treatment period, each animal was sedated with propofol and administered a bolus of fenfluramine (5 mg/kg). Fenfluramine causes the release of serotonin proportional to endogenous availability and in turn, serotonin stimulates the secretion of prolactin. Therefore, serum prolactin concentrations reflect endogenous serotonin. Fenfluramine significantly increased serotonin/prolactin in all groups (p < .0001). Fenfluramine-induced serotonin/prolactin in the T-treated group was significantly higher than the other groups (p < .0001). Castration partially reduced the serotonin/prolactin response and Letrozole partially blocked the effect of T. Complete inhibition of aromatase with ATD, a noncompetitive inhibitor, significantly and similarly reduced the fenfluramine-induced serotonin/prolactin response in the presence or absence of DHT. Neither aggressive behavior nor yawning (indicators of androgen activity) correlated with serotonin/prolactin, but posited aromatase activity correlated significantly with prolactin (p < .0008; r2 = 0.95). In summary, androgens induced aggressive behavior but they did not regulate serotonin. Altogether, the data suggest that aromatase activity supports serotonin production and that androgens increase aggression by another mechanism. (PsycINFO Database Record (c) 2013 APA, all rights reserved).
Behavioral Neuroscience 03/2013; · 2.62 Impact Factor
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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 60 days, 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 60 days (SS). This study examines serotonin-related gene expression in monkeys with different sensitivity to stress and exposed to 5 days of moderate stress. Monkeys were first characterized as HSR, MSR or SS. After resumption of menstrual cycles, each monkey was re-stressed for 5 days 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 digoxygenin-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 5 days 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.
Progress in Neuro-Psychopharmacology and Biological Psychiatry 01/2013; · 3.25 Impact Factor
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ABSTRACT: Communication between the serotonin system and the CRF system plays a pivotal role in the mediation of stress and stress reactivity. CRF appears to be inhibitory of serotonin neurotransmission through the CRF receptor type 1 (CRF-R1). Serotonin neurons also detect the urocortins, which are thought to be anxiolytic. Components of the CRF system in the serotonergic dorsal raphe region were examined in macaques that were ovary-intact or ovariectomized for 3 years living in a relatively natural environment. Female Japanese macaques (Macaca fuscata) were ovariectomized or tubal-ligated (n=5/group) and returned to their natal troop for 3 years. Quantitation of (1) CRF innervation of the serotonergic dorsal raphe, (2) CRF-Receptor type 1 (CRF-R1) in the dorsal raphe, (3) Urocortin 1 (UCN1) cells near the Edinger-Westfal nucleus and (4) UCN1 axons, was obtained with immunocytochemical staining and image analysis. There was no statistical difference in CRF axonal staining in the dorsal raphe, or in UCN1 axonal staining near the dorsal raphe. However, the average number of detectable UCN1 postive cells was significantly lower in the Ovx group than in the Intact group (p=0.003). Average CRF-R1 positive pixel number and positive cell number were significantly higher in the Ovx group than in the Intact group (p=0.005 and 0.02, respectivly). The higher expression of CRF-R1 and lower expression of UCN1 in the Ovx group indicates they may be more vulnerable to stress. The greater expression of CRF-R1 could cause a greater inhibition of serotonin upon a stress-induced increase in CRF as well.
Brain research 10/2012; · 2.46 Impact Factor
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ABSTRACT: The purpose of this study was to investigate the protective effects of the mitochondria-targeted antioxidant catalase (MCAT) and lifespan extension in mice that express amyloid beta (Aβ). Using immunoblotting and immunostaining analyses, we measured the production of full-length amyloid precursor protein (APP), soluble APPα, C-terminal fragments CTF99 and CTF83, monomeric and oligomeric Aβ, Aβ deposits and beta site amyloid precursor protein cleaving enzyme 1 (BACE1), in different stages of disease progression in MCAT/AβPP and AβPP mice. Using quantitative reverse transcriptase polymerase chain reaction and immunostaining analyses, we studied the expression of catalase, BACE1, the Alzheimer's disease (AD) markers, synaptophysin, APP, neprilysin, insulin-degrading enzyme and transthyretin in MCAT, AβPP, MCAT/AβPP and wild-type (WT) mice. Using the high pressure liquid chromatography analysis of 8-hydroxy-2-deoxyguanosine, we measured oxidative DNA damage in the cerebral cortical tissues from MCAT, AβPP, MCAT/AβPP and WT mice. We found that the AβPP transgenic mice that carried the human MCAT gene lived 5 months longer than did the AβPP mice. We also found that the overexpression of MCAT in the brain sections from the MCAT/AβPP transgenic mice significantly correlated with a reduction in the levels of full-length APP, CTF99, BACE1, Aβ levels (40 and 42), Aβ deposits and oxidative DNA damage relative to the brain sections from the AβPP mice. Interestingly, we found significantly increased levels of soluble APPα and CTF83 in the MCAT/AβPP mice, relative to the AβPP mice. These data provide direct evidence that oxidative stress plays a primary role in AD etiopathology and that in MCAT mice express Aβ, MCAT prevents abnormal APP processing, reduces Aβ levels and enhances Aβ-degrading enzymes in mice at different ages, corresponding to different stages of disease progression. These findings indicate that mitochondria-targeted molecules may be an effective therapeutic approach to treat patients with AD.
Human Molecular Genetics 04/2012; 21(13):2973-90. · 7.64 Impact Factor
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ABSTRACT: Dendritic spines are the elementary structural units of neural plasticity. In a model of hormone replacement therapy (HT), we sought to determine the effect of estradiol (E) and progesterone (P) on gene expression related to synapse assembly in a laser-captured preparation enriched for serotonin neurons from rhesus macaques. Microarray analysis was conducted (n = 2 animals/treatment), and the results were confirmed for pivotal genes with qRT-PCR on additional laser-captured material (n = 3 animals/treatment). Ovariectomized rhesus macaques were treated with placebo, E, or E + P via Silastic implants for 1 month. The midbrain was obtained, sectioned, and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an arcturus laser dissection microscope (Pixel II). RNA from laser-captured serotonin neurons was hybridized to Rhesus Affymetrix GeneChips for screening purposes. There was a twofold or greater change in the expression of 63 probe sets in the cell adhesion molecule (CAM) category, and 31 probe sets in the synapse assembly category were similarly altered in E- and E + P-treated animals. qRT-PCR assays showed that E treatment induced a significant increase in ephrin receptor A4 (EPHA4) and in integrin A8 (ITGA8) but not in ephrin receptor B4 (EPHB4) or integrin B8 (ITGB8) expression. E also increased expression of cadherin 11 (CDH11), neuroligin 3 (NLGN3), neurexin 3 (NRXN3), syndecan 2 (SCD2), and neural cell adhesion molecule (NCAM) compared with placebo. Supplemental P treatment suppressed E-induced gene expression. In summary, ovarian steroids target gene expression of adhesion molecules in serotonin neurons that are important for synapse assembly.
Journal of Neuroscience Research 03/2012; 90(7):1324-34. · 2.74 Impact Factor
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ABSTRACT: We have demonstrated marked differences in the neurobiology of the serotonin system between stress-sensitive (SS) and stress-resilient (SR) cynomolgus macaques characterized in a model of stress-induced amenorrhea, also called functional hypothalamic amenorrhea (FHA). Dysfunction of the serotonin system in SS monkeys suggested that administration of a selective serotonin reuptake inhibitor (SSRI) might correct FHA. This study examines the effect of escitalopram (CIT) administration to SS and SR monkeys on corticotrophin-releasing factor (CRF) receptor 1 (CRF-R1) and CRF receptor 2 (CRF-R2) gene expression in the serotonin cell body region of the midbrain dorsal raphe. CRF-R1 was not significantly different between groups. There was a significant effect of treatment and a significant interaction between treatment and stress sensitivity on the average CRF-R2-positive pixel area (P < .004 and P < .006, respectively) and on the average number of CRF-R2-positive cells (P < .023 and P < .025, respectively). CIT significantly increased CRF-R2-positive pixel area and cell number in the SS group (pixel area P < .001; cell number P < .01; Bonferoni) but not in the SR group. In summary, CIT administration tended to decrease CRF-R1, but the small animal number precluded significance. CIT administration significantly increased CRF-R2 only in SS animals. These data suggest that the administration of CIT reduces anxiogenic components and increases anxiolytic components of the CRF system in the midbrain serotonin network, which in turn leads to improved ovarian function. Moreover, these data raise the possibility that SSRIs may be effective in the treatment of stress-induced infertility.
Reproductive sciences (Thousand Oaks, Calif.) 03/2012; 19(6):623-32. · 2.31 Impact Factor
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ABSTRACT: Dendritic spines are the elementary structural units of neuronal plasticity and their proliferation and stabilization involve components of glutamate neurotransmission. In a model of hormone replacement therapy (HT), we sought the effect of estradiol (E) and progesterone (P) on gene expression related to glutamate neurotransmission in a laser captured preparation enriched for serotonin neurons from rhesus macaques. Microarray analysis was conducted (n=2 animals/treatment) and then confirmed for pivotal genes with qRT-PCR on additional laser captured material (n=3 animals/treatment). Ovariectomized rhesus macaques were treated with either placebo, E or E+P via Silastic implants for 1month prior to euthanasia. The midbrain was obtained, sectioned and immunostained for TPH. TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (Pixel II). RNA from laser captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips for screening purposes. There was a 2-fold or greater change in the expression of 28 probe sets related to glutamate processes in E and E+P treated animals. Quantitative (q) RT-PCR was conducted for 11 genes with a custom Taqman PCR array containing monkey specific primers and analyzed with ANOVA followed by Bonferroni's test. The log of the relative expression values indicated that in general, the responses to E and E+P were similar. Comparison of the relative expression or log relative expression in Ovx-controls to combined E and E+P treated groups with t-tests showed a significant increase in AMPA1 (GRIA1), AMPA2 (GRIA2), AMPA4 (GRIA4), NMDA2a (GRIN2A), metabotrophic glutamate receptor (GRM1), glutamine synthetase (GLUL), glutamate dehydrogenase (GLUD), glutamate cysteine ligase modifier subunit (GCLM), the glutamate transporter 2 (SLC1A2) and the glutamate transporter 3 (SLC1A3) with steroid treatment. There was no effect of steroid treatment on gene expression of the glutamate cysteine ligase catalytic subunit (GCLC). These data suggest that ovarian steroids target gene expression of ionotrophic and metabotrophic glutamate receptors in serotonin neurons. These receptors are present on dendritic spines and are necessary for spine maturation. The mRNAs coding for glutamate-related enzymes and transporters are likely derived from astrocytes or glutamate-containing terminals. Their induction by ovarian steroids indicates a complex upregulation of multiple components in the glutamate cycle and antioxidation, in addition to spine proliferation.
Molecular and Cellular Neuroscience 11/2011; 49(3):251-62. · 3.66 Impact Factor
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P Hemachandra Reddy,
Raghav Tripathi,
Quang Troung,
Karuna Tirumala,
Tejaswini P Reddy,
Vishwanath Anekonda,
Ulziibat P Shirendeb,
Marcus J Calkins, Arubala P Reddy,
Peizhong Mao,
Maria Manczak
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ABSTRACT: Synaptic pathology and mitochondrial oxidative damage are early events in Alzheimer's disease (AD) progression. Loss of synapses and synaptic damage are the best correlates of cognitive deficits found in AD patients. Recent research on amyloid beta (Aβ) and mitochondria in AD revealed that Aβ accumulates in synapses and synaptic mitochondria, leading to abnormal mitochondrial dynamics and synaptic degeneration in AD neurons. Further, recent studies using live-cell imaging and primary neurons from amyloid beta precursor protein (AβPP) transgenic mice revealed reduced mitochondrial mass, defective axonal transport of mitochondria and synaptic degeneration, indicating that Aβ is responsible for mitochondrial and synaptic deficiencies. Tremendous progress has been made in studying antioxidant approaches in mouse models of AD and clinical trials of AD patients. This article highlights the recent developments made in Aβ-induced abnormal mitochondrial dynamics, defective mitochondrial biogenesis, impaired axonal transport and synaptic deficiencies in AD. This article also focuses on mitochondrial approaches in treating AD, and also discusses latest research on mitochondria-targeted antioxidants in AD. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.
Biochimica et Biophysica Acta 10/2011; 1822(5):639-49. · 4.66 Impact Factor
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ABSTRACT: 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.
Journal of chemical neuroanatomy 06/2011; 41(4):200-18. · 1.75 Impact Factor
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ABSTRACT: The purpose of our study was to determine the relationship between mutant huntingtin (Htt) and mitochondrial dynamics in the progression of Huntington's disease (HD). We measured the mRNA levels of electron transport chain genes, and mitochondrial structural genes, Drp1 (dynamin-related protein 1), Fis1 (fission 1), Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), Opa1 (optric atrophy 1), Tomm40 (translocase of outermembrane 40) and CypD (cyclophilin D) in grade III and grade IV HD patients and controls. The mutant Htt oligomers and the mitochondrial structural proteins were quantified in the striatum and frontal cortex of HD patients. Changes in expressions of the electron transport chain genes were found in HD patients and may represent a compensatory response to mitochondrial damage caused by mutant Htt. Increased expression of Drp1 and Fis1 and decreased expression of Mfn1, Mfn2, Opa1 and Tomm40 were found in HD patients relative to the controls. CypD was upregulated in HD patients, and this upregulation increased as HD progressed. Significantly increased immunoreactivity of 8-hydroxy-guanosine was found in the cortical specimens from stage III and IV HD patients relative to controls, suggesting increased oxidative DNA damage in HD patients. In contrast, significantly decreased immunoreactivities of cytochrome oxidase 1 and cytochrome b were found in HD patients relative to controls, indicating a loss of mitochondrial function in HD patients. Immunoblotting analysis revealed 15, 25 and 50 kDa mutant Htt oligomers in the brain specimens of HD patients. All oligomeric forms of mutant Htt were significantly increased in the cortical tissues of HD patients, and mutant Htt oligomers were found in the nucleus and in mitochondria. The increase in Drp1, Fis1 and CypD and the decrease in Mfn1 and Mfn2 may be responsible for abnormal mitochondrial dynamics that we found in the cortex of HD patients, and may contribute to neuronal damage in HD patients. The presence of mutant Htt oligomers in the nucleus of HD neurons and in mitochondria may disrupt neuronal functions. Based on these findings, we propose that mutant Htt in association with mitochondria imbalance and mitochondrial dynamics impairs axonal transport of mitochondria, decreases mitochondrial function and damages neurons in affected brain regions of HD patients.
Human Molecular Genetics 02/2011; 20(7):1438-55. · 7.64 Impact Factor
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ABSTRACT: The purpose of this study was to determine the neurotoxicity of two commonly used herbicides: picloram and triclopyr and the neuroprotective effects of the mitochondria-targeted antioxidant, SS31. Using mouse neuroblastoma (N2a) cells and primary neurons from C57BL/6 mice, we investigated the toxicity of these herbicides, and protective effects of SS1 peptide against picloram and triclopyr toxicity. We measured total RNA content, cell viability and mRNA expression of peroxiredoxins, neuroprotective genes, mitochondrial-encoded electron transport chain (ETC) genes in N2a cells treated with herbicides and SS31. Using primary neurons from C57BL/6 mice, neuronal survival was studied in neurons treated with herbicides, in neurons pretreated with SS31 plus treated with herbicides, neurons treated with SS31 alone, and untreated neurons. Significantly decreased total RNA content, and cell viability in N2a cells treated with picloram and triclopyr were found compared to untreated N2a cells. Decreased mRNA expression of neuroprotective genes, and ETC genes in cells treated with herbicides was found compared to untreated cells. Decreased mRNA expression of peroxiredoxins 1-6 in N2a cells treated with picloram was found, suggesting that picloram affects the antioxidant enzymes in N2a cells. Immunofluorescence analysis of primary neurons revealed that decreased neuronal branching and degenerating neurons in neurons treated with picloram and triclopyr. However, neurons pretreated with SS31 prevented degenerative process caused by herbicides. Based on these results, we propose that herbicides--picloram and triclopyr appear to damage neurons, and the SS31 peptide appears to protect neurons from herbicide toxicity.
International Journal of Environmental Research and Public Health 01/2011; 8(1):203-21. · 1.61 Impact Factor
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ABSTRACT: This article reviews the role of amyloid-beta (Abeta) and mitochondria in synaptic damage and cognitive decline found in patients with Alzheimer's disease (AD). Recent molecular, cellular, animal model, and postmortem brain studies have revealed that Abeta and mitochondrial abnormalities are key factors that cause synaptic damage and cognitive decline in AD. Abeta is reported to accumulate in subcellular compartments and to impair the normal function of neurons in AD patients. Further, recent studies using biochemical methods and electron microscopy have revealed that the accumulation of Abeta at nerve terminals affect synaptic activities, including the release of neurotransmitters and synaptic vesicles. Recent studies of the relationship between mitochondria and Abeta in AD patients suggest that in mitochondria, structural changes caused by Abeta result in increased mitochondrial fragmentation, decreased mitochondrial fusion, mitochondrial dysfunction, and synaptic damage. This paper discusses the latest research on Abeta, mitochondria, age-dependent factors of AD in the brain, and synaptic damage in AD. This paper also briefly discusses potential mitochondrial therapeutics in the treatment of patients with AD.
Journal of Alzheimer's disease: JAD 04/2010; 20 Suppl 2:S499-512. · 3.74 Impact Factor
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ABSTRACT: The purpose of our study was to investigate the effects of the mitochondria-targeted antioxidants, MitoQ and SS31, and the anti-aging agent resveratrol on neurons from a mouse model (Tg2576 line) of Alzheimer's disease (AD) and on mouse neuroblastoma (N2a) cells incubated with the amyloid-beta (Abeta) peptide. Using electron and confocal microscopy, gene expression analysis, and biochemical methods, we studied mitochondrial structure and function and neurite outgrowth in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta. In N2a cells only incubated with the Abeta, we found increased expressions of mitochondrial fission genes and decreased expression of fusion genes and also decreased expression of peroxiredoxins. Electron microscopy of the N2a cells incubated with Abeta revealed a significantly increased number of mitochondria, indicating that Abeta fragments mitochondria. Biochemical analysis revealed that function is defective in mitochondria. Neurite outgrowth was significantly decreased in Abeta-incubated N2a cells, indicating that Abeta affects neurite outgrowth. However, in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta, abnormal expression of peroxiredoxins and mitochondrial structural genes were prevented and mitochondrial function was normal; intact mitochondria were present and neurite outgrowth was significantly increased. In primary neurons from amyloid-beta precursor protein transgenic mice that were treated with MitoQ and SS31, neurite outgrowth was significantly increased and cyclophilin D expression was significantly decreased. These findings suggest that MitoQ and SS31 prevent Abeta toxicity, which would warrant the study of MitoQ and SS31 as potential drugs to treat patients with AD.
Journal of Alzheimer's disease: JAD 01/2010; 20 Suppl 2:S609-31. · 3.74 Impact Factor
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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 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.
Neuroscience 09/2009; 164(2):676-91. · 3.38 Impact Factor
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ABSTRACT: The serotonin neurons of the dorsal and medial raphe nuclei project to all areas of the forebrain and play a key role in mood disorders. Hence, any loss or degeneration of serotonin neurons could have profound ramifications. In a monkey model of surgical menopause with hormone replacement and no neural injury, E and P decreased gene expression in the dorsal raphe nucleus of c-jun n-terminal kinase (JNK1) and kynurenine mono-oxygenase (KMO) that promote cell death. In concert, E and P increased gene expression of superoxide dismutase (SOD1), VEGF, and caspase inhibitory proteins that promote cellular resilience in the dorsal raphe nucleus. Subsequently, we showed that ovarian steroids inhibit pivotal genes in the caspase-dependent and caspase-independent pathways in laser-captured serotonin neurons including apoptosis activating factor (Apaf1), apoptosis-inducing factor (AIF) and second mitochondria-derived activator of caspases (Smac/Diablo). SOD1 was also increased specifically in laser-captured serotonin neurons. Examination of protein expression in the dorsal raphe block revealed that JNK1, phosphoJNK1, AIF and the translocation of AIF from the mitochondria to the nucleus decreased with hormone therapy, whereas pivotal execution proteins in the caspase pathway were unchanged. In addition, cyclins A, B, D1 and E were inhibited, which would prevent re-entry into the cell cycle and catastrophic death. These data indicated that in the absence of gross injury to the midbrain, ovarian steroids inhibit the caspase-independent pathway and cell cycle initiation in serotonin neurons. To determine if these molecular actions prevented cellular vulnerability or death, we examined DNA fragmentation in the dorsal raphe nucleus with the TUNEL assay (terminal deoxynucleotidyl transferase nick end labeling). Ovarian steroids significantly decreased the number of TUNEL-positive cells in the dorsal raphe. Moreover, TUNEL staining prominently colocalized with TPH immunostaining, a marker for serotonin neurons. In summary, ovarian steroids increase the cellular resilience of serotonin neurons and may prevent serotonin neuron death in women facing decades of life after menopause. The survival of serotonin neurons would support cognition and mental health.
Frontiers in Neuroendocrinology 05/2009; 30(2):212-38. · 11.43 Impact Factor
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ABSTRACT: The rhesus monkey embryonic stem cell line 366.4 differentiates into serotonin neurons. We examined the genetic cascade during differentiation and compared ESC-derived serotonin neurons to adult monkey serotonin neurons. RNA was extracted from ESC colonies, embryoid bodies (EBs), neurospheres in selection (N1) and proliferation stages (N2), differentiated serotonin neurons (N3) and from laser captured (LC) serotonin neurons of spayed female macaques treated with placebo, estrogen (E), progesterone (P) or E+P. The RNA was labeled and hybridized to Rhesus Monkey Affymetrix Gene Chips (n=1 per stage and 2 per animal treatment). Gene expression was examined with GeneSifter software. 545 genes that were related to developmental processes showed a threefold or greater change between stages. TGFb, Wnt, VEGF and Hedgehog signaling pathways showed the highest percent of probe set changes during differentiation. Genes in the categories (a) homeobox binding and transcription factors, (b) growth factors and receptors, (c) brain and neural specific factors and (d) serotonin specific factors are reported. Pivotal genes were confirmed with quantitative RT-PCR. In the serotonin developmental cascade, FGFR2 was robustly expressed at each stage. GATA3 was robustly expressed in EBs. Sonic hedgehog (Shh), PTCH (Shh-R) and Fev1 transcription factor expression coincided with the induction of serotonin specific marker genes during N1-selection. A majority of the examined genes were expressed in adult serotonin neurons. However, in the ESC-derived neurons, there was significant over-representation of probe sets related to cell cycle, axon guidance & dorso-ventral axis formation. This analysis suggests that the 366.4 cell line possesses cues for serotonin differentiation at early stages of differentiation, but that ESC-derived serotonin neurons are still immature.
Gene Expression Patterns 11/2008; 9(2):94-108. · 2.02 Impact Factor
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ABSTRACT: We sought the effect of estradiol (E) and progesterone (P) on survival gene expression in laser captured serotonin neurons and in the dorsal raphe region of monkeys with cDNA array analysis. Spayed rhesus macaques were treated with either placebo, E or E + P via Silastic implant for 1 month prior to killing. First, RNA from a small block of midbrain containing the dorsal raphe was hybridized to Rhesus Gene Chips (n = 3/treatment). There was a significant change in 854 probe sets with E +/- P treatment (anova, p < 0.05); however, only 151 probes sets exhibited a twofold or greater change. Twenty-five genes related to cell survival changed significantly. The expression of vascular endothelial growth factor, superoxide dismutase (SOD1), and the caspase inhibitor, BIRC4, was confirmed with quantitative RT-PCR. Then, RNA from laser captured serotonin neurons (n = 2/treatment) was hybridized to Rhesus Gene Chips. There was a significant change in 744 probe sets, but 10 493 probe sets exhibited a twofold or greater change. Pivotal changes in apoptosis and cell cycle pathways included twofold or greater increases in SOD1, IkappaBalpha, Fas apoptotic inhibitory molecule, fibroblast growth factor-receptor 2 (FGFR2), neurotrophic tyrosine kinase receptor 2 (NTRK2), phosphoinositide-3-kinase (p85 subunit), cyclic AMP dependent protein kinase (PKA) (catalytic subunit), calpain 2, and ataxia telangectasia mutated (ATM). Twofold or greater decreases occurred in TNF receptor interacting serine-threonine kinase 1 (RIP1), BH3 interacting domain death agonist (BID), apoptotic peptidase activating factor 1 (Apaf1), caspase recruitment domain 8 (CARD8), apoptosis inducing factor (AIF), Diablo and Cyclins A, B, D, and E. The regulation of SOD1, calpain 2, Diablo, and Cyclin D was confirmed with quantitative RT-PCR (n = 3/treatment). The data indicate that ovarian steroids target the cytokine-signaling pathway, caspase-dependent and -independent pathways and cell cycle proteins to promote serotonin neuron survival.
Journal of Neurochemistry 05/2008; 105(4):1129-43. · 4.06 Impact Factor
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ABSTRACT: Estrogen and progesterone act on gene and protein expression in serotonin neurons in a manner that suggests serotonin neurotransmission should increase. However, measurement of extracellular serotonin in macaques was lacking. Elevated prolactin secretion can be an indicator of increased serotonergic function and prolactin is increased by combined estrogen and progesterone treatment. We examined extracellular serotonin by microdialysis in a well-characterized macaque model of steroid-induced prolactin secretion. Monkeys were fitted with 2 guide tubes directed to the arcuate nucleus of the hypothalamus. Samples (75 microl/15-minute interval) were obtained via a tether-swivel device through sample lines into an adjoining room. Serotonin was measured with a modified commercial enzyme linked immunoassay (ELISA) kit. Fenfluramine infused through the probe (300 microM for 2 h; n=2 trials) or administered intravenously (2.5 mg/kg; n=2 trials) caused a marked increase in extracellular serotonin and verified the efficacy of the procedure. Three monkeys were maintained with an estrogen implant for 2 weeks. Each monkey was injected with 20 mg of progesterone s.c. in oil at 1500 h; microdialysis was initiated the next morning and samples were obtained for 24 h. There was a significant increase in serotonin between 40 and 43 h after the progesterone injection (P<0.001, ANOVA). Serotonin averaged 59+/-1 pg/sample from 18-30 h post-progesterone injection, and averaged 76+/-2 pg/sample from 30-48 h post-progesterone injection (P<0.0001; t-test). Since the increase in serotonin is delayed by approximately 40 h after progesterone-injection, we speculate that the action of progesterone may involve either nuclear progestin receptors or membrane progestin receptors.
European Journal of Pharmacology 02/2007; 555(1):67-75. · 2.52 Impact Factor
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ABSTRACT: The expressions of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC) were assessed in brain tissue collected from nonstressed female cynomolgus monkeys previously categorized as highly stress resilient (HSR), medium stress resilient (MSR), or stress sensitive (SS) with respect to stress-induced anovulation.
In situ hybridization and quantitative image analysis was used to measure mRNAs coding for CRH in the hypothalamic paraventricular nucleus (PVN) and thalamic center median-subfascicular complex (CM-Sf). Then, CRH neurons in the PVN were immunostained and the area of immunostaining was measured. Also, CRH fibers were immunostained in the central nucleus of the amygdala and the area of immunostaining was obtained. Finally, POMC mRNA expression was characterized in the hypothalamic infundibular nucleus. The groups were compared with ANOVA and Student-Newman-Keul's (SNK) post hoc comparison.
CRH mRNA was significantly elevated in the caudal PVN in the MSR and SS animals compared to HSR animals (p < 0.05, SNK). There was a significant increase in average and total CRH-positive area in the MSR and SS groups compared to the HSR group (p < 0.05, SNK). There was also a significant increase in CRH volume in the MSR and SS groups compared to the HSR group (p < 0.05, SNK). In the CM-Sf, the average CRH optical density was significantly higher in the MSR and SS groups than in the HSR group (p < 0.05, SNK). In the central nucleus of the amygdala, the area of CRH fiber staining was significantly higher in the SS group than in the MSR or HSR groups (p < 0.05, SNK). There was no difference between the groups in POMC mRNA expression in the mediobasal hypothalamus.
Macaques that exhibit immediate suppression of reproductive function upon stress are considered stress sensitive. These animals have elevated CRH in the hypothalamus and limbic structures, which may play a role in suppressing the hypothalamic-gonadal axis upon stress initiation.
Neuroendocrinology 01/2007; 86(4):277-88. · 2.38 Impact Factor
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ABSTRACT: Nuclear factor kappa B (NFkappaB) is a transcription factor that activates gene expression in response to proinflammatory cytokines, and elevated cytokines are associated with depression, which has a serotonergic component. We questioned (1) whether serotonin neurons contain NFkappaB, (2) whether NFkappaB detection with immunocytochemistry is changed in the dorsal raphe nucleus (DRN) by ovarian hormone treatment and (3) whether ovarian hormones regulate midbrain NFkappaB gene or protein expression.
Monkeys were spayed and treated with placebo, estrogen (E), progesterone (P) or E+P for 1 month (n = 4 animals/treatment group), and the midbrain was harvested for immunocytochemistry and stereology. An antibody that detects nuclear location-specific (NLS)-NFkappaB p65 was applied, and the numbers of NLS-NFkappaB-immunopositive cells were counted in 9 sections of the DRN. Additional monkeys were used for Western blot analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) for NFkappaB p65.
In placebo-treated macaques, neurons were double-immunostained for serotonin and nuclear NFkappaB p65 throughout the DRN. The mean total number of NFkappaB-positive cells equalled 2178 (and standard error of the mean [SEM] 129) in the placebo group, 1631 (SEM 221) in the E-treated group, 2314 (SEM 186) in the P-treated group and 1162 (SEM 100) in the E+P-treated group (analysis of variance p = 0.003). The E-treated and E+P-treated groups had a significantly lower density of cells stained positive for NFkappaB than the placebo or P-treated groups (post hoc). Unmasking of NLS-NFkappaB immunostaining in the DRN revealed dense immunostaining in the cytoplasm of large dorsal raphe neurons. There was no difference between treatment groups in the amount of NFkappaB p65 detected by Western blot or in the relative expression of NFkappaB p65 mRNA with quantitative RT-PCR.
These observations are consistent with the notion that gene and protein expression of NFkappaB are constitutive but that ovarian hormones can decrease the nuclear location of NFkappaB in dorsal raphe neurons and, thereby, decrease the ability of NFkappaB to drive gene expression in response to cytokines.
Journal of psychiatry & neuroscience: JPN 04/2006; 31(2):105-14. · 5.34 Impact Factor
