Central resistance to the actions of insulin and leptin is strongly associated with obesity and type 2 diabetes mellitus (T2DM). These anorexigenic hormones modulate one another’s actions at the neuronal level. To investigate the cellular events underlying the effect of insulin resistance on leptin modulation of hypothalamic neurons, a neuronal cell model was established. The rHypoE-19 cell line expresses the insulin and leptin receptors alongside a complement of signaling molecules rendering it an appropriate model to study the molecular events underlying leptin and insulin crosstalk. Hyperinsulinemia was used to induce insulin resistance and leptin regulation of the rHypoE-19 neurons was analyzed prior to and following the induction of insulin resistance. It was found that the attenuation of insulin signal transduction affects leptin signaling and transcriptional modulation of the rHypoE-19 neurons. These studies will ultimately lend itself to an improved understanding of the complex cellular events that accompany neuronal hormone resistance.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism.

A series of eight experiments was conducted to investigate the requirement for thyroid hormones in neuroendocrine processes which lead to the seasonally anoestrous state in red deer hinds. The first two experiments used thyroidectomized, ovariectomized, oestradiol-treated hinds which received various thyroid hormone replacement treatments (n=5 per group) to investigate the timing and dose-responsiveness of thyroid hormones in bringing about seasonal oestradiol-induced suppression of plasma LH concentration. A significant seasonal decline in mean plasma LH concentration during September (coinciding with the onset of anoestrus in entire cycling hinds in New Zealand) was observed in all thyroidectomized hinds in both experiments regardless of T₄ or T₃ treatment. When oestradiol implants were removed in November or December, mean plasma LH concentrations increased significantly in all but one of hinds in which T₄ had been administered at very low doses by subcutaneous implants, and mean plasma LH concentrations and LH pulse amplitude increased in approximately half of hinds administered T₃ at varying doses by subcutaneous injections over a one-week period in October. These results suggested that thyroid hormones are not required for steroid-dependent reproductive suppression, but could possibly play a role in steroid-independent suppression of LH secretion. Because problems were encountered in delivering appropriate doses of thyroid hormones in both experiments, further confirmation of these findings was required. Therefore in the next experiment the role of thyroid gland secretions was examined in euthyroid (n=5) and thyroidectomized (n=4) ovariectomized hinds treated with oestradiol implants. These implants were removed for about one month on three occasions to examine the effect of thyroidectomy on steroid-independent control of seasonal LH secretion. During the non-breeding season basal and GnRH-induced plasma LH concentrations declined in all hinds in the presence of oestradiol, but returned to breeding season levels when oestradiol was withdrawn in November. In a concurrent experiment, thyroidectomy of ovary-entire hinds (n=7) during the breeding season prevented the cessation of oestrous cyclicity in spring; this was in contrast to oestrous cyclicity in T₄replaced (n=4) or euthyroid control (n=5) hinds which ceased to occur in early September. Collectively, these results indicate that thyroid hormones are required for the termination of the breeding season in cycling red deer hinds and that this action occurs via steroid-independent neuroendocrine pathways. Two experiments were conducted using neurotransmitter receptor agonists and antagonists to identify neural pathways in the brain which mediate LH suppression by oestradiol and by steroid-independent mechanisms, and to test if the thyroid gland is required for activation of these pathways during the non-breeding season. It was concluded from the lack of plasma LH responses to dopaminergic and opioidergic agonists and antagonists in ovariectomized and ovariectomized, thyroidectomized hinds (n=5) that neural pathways involving dopamine-D₂receptors do not mediate oestradiol-induced seasonal suppression of plasma LH concentrations, and neither dopaminergic or opioid neural pathways mediate non-steroidal suppression of plasma LH concentrations. However preliminary evidence was obtained for a stimulatory role of serotonergic neural pathways in controlling LH secretion. Another experiment was conducted to identify when the steroid-independent mechanisms which suppress LH concentrations during the non-breeding season are responsive to thyroid hormones. T₄treatment at the beginning of or during the non breeding season was effective in bringing about suppression of plasma LH concentration in thyroidectomized, ovariectomized hinds (n=5 per group), but this action of thyroid hormones did not occur during the breeding season. These results show that the steroid-independent mechanisms which contribute to seasonal suppression of plasma gonadotrophin concentrations require thyroid hormones to be present only from around the time of the end of the breeding season for their normal expression, and they remain responsive to thyroid hormones after this period. Lastly, the feasibility of achieving out-of-season breeding using thyroidectomized hinds (n=9) was evaluated by comparing oestrous behaviour, ovulation and pregnancy rates to those of euthyroid control hinds (n=7) following synchronization of oestrous cycles. There was a non-significant trend for a greater occurrence of oestrous behaviour and ovulation in thyroidectomized hinds compared with euthyroid controls during the non-breeding season, but the pregnancy rate following out-of season mating with a thyroidectomized stag was low, suggesting that a side effect of thyroidectomy may be impaired fertility. Six out-of-season pregnancies were obtained from eight matings, however because three of these pregnancies occurred in euthyroid control hinds no improvement in out-of-season reproductive performance could be attributed to thyroidectomy. It is likely that if the actions of the thyroid glands are to be exploited as a tool for achieving out-of-season breeding in this species, techniques will have to be developed for specifically blocking or overcoming the effects of thyroid hormones on the reproductive neuroendocrine centres without causing general hypothyroidism and its associated side-effects.

Maintenance of normal health requires an intact stress system capable of mounting the metabolic, autonomic, behavioural and motor responses required for coping with or avoiding physiological and pathological challenges. The neuroendocrine component of this response principally involves the hypothalamic-pituitary-adrenal (HPA) and sympatho-adrenomedullary (SAM) axes. Impaired regulation of these axes has been implicated in the pathogenesis and expression of numerous disease states, however, it has proved very difficult to reproducibly activate the HPA and SAM axes and no single test exists that can reliably and safely be used to study these systems in man. Carbon dioxide (CO2) is the principal regulator of respiration, acid-base balance and behavioural-state arousal in humans. Paradigms of CO2 inhalation have been used in psychiatric research to investigate panic and anxiety disorders, but evaluation of other components of the stress response to CO2 has not previously been performed. I hypothesised that a single breath of 35% CO2 would be a simple and reliable tool for the evaluation of the stress response in humans. A single breath of four doses of CO2 (5%, 25%, 35% and 50%) was administered to 9 healthy volunteers in a randomised, single blind fashion. Subjective symptoms of anxiety increased in a dose-dependent manner. Inhalation of a single breath of 35% CO2 stimulated significant ACTH (p = 0.006), noradrenaline (p < 0.0001), cortisol (p = 0.02) and prolactin (p = 0.002) release. It also provoked an acute pressor response and an associated bradycardia (p < 0.0001 for both). No significant habituation of psychological, HPA or cardiovascular responses was seen when this dose was repeated after one week (n = 10) or 6 months (n = 5). It was apparent that a single breath of 35% CO2 reliably and safely produced SAM and HPA axis activation and further studies were then undertaken to assess the mechanism by which the observed responses occurred and its potential clinical implications. Administration of naltrexone (an opiate antagonist) to 10 normal volunteers disinhibited the HPA axis (p < 0.0004), whilst administration of metyrapone (a cortisol synthesis inhibitor) significantly reduced baseline cortisol (p < 0.03) levels. However, this alteration in HPA axis activity had no effect on either cardiovascular or psychological responses. Further, in a study of 8 breastfeeding mothers (a state associated with physiological suppression of the HPA axis) suckling significantly reduced plasma cortisol levels compared with control (p = 0.002) and bottle-feeders (p = 0.003). Despite this cortisol, systolic blood pressure (SBP), heart rate and psychological responses to 35% CO2 were not affected

Aromatase is an enzyme that converts androgens to estrogens. In teleosts, brain aromatase, also known as aromatase B (cy19a1b), is only expressed in radial glial cells (RGCs). This is in contrast to aromatase A, which is expressed in gonads. Estrogens such as estradiol (E2) modulate neurogenesis in the adult teleost brain. Recent studies show that E2 also differentially regulates aromatase B expression in goldfish RGCs. As a result, teleost RGCs are suggested to be involved in regulating neurogenesis. In addition, aromatase B expression in goldfish RGC is under the control of dopamine suggesting that neurons and neurotransmitters can regulate RGC function. Interestingly, goldfish RGC transcriptome data shows the expression of one group of metabotropic glutamate receptors (mGluRs), group III mGluRs, which suggests that glutamate may affect RGC function. In this thesis, I present my findings regarding potential glutamatergic regulation of RGCs. Firstly, I investigated the distribution of glutamatergic synaptic vesicles and RGCs in the female goldfish forebrain. Double-staining immunohistochemistry shows that vesicular glutamate transporter (vGLUT) 1/2-labelled glutamatergic synaptic vesicles are in close anatomical proximity to aromatase B-labelled RGCs, which suggests potential regulation of RGCs by glutamate. Glutamatergic regulation of cyp19a1b, cyclin D1 (ccnd1), cyclin A2 (ccna2), mGluR6b (grm6b), mGluR7 (grm7), and mGluR8b (grm8b) expression in cultured adult female goldfish RGCs was also examined. Results from pharmacological manipulations and qPCR data analysis show that selective activation of group III mGluRs decreased cyp19a1b, ccnd1, and ccna2 mRNA via inhibition of cAMP/PKA signalling. Furthermore, grm7 mRNA is positively regulated by cAMP-dependent signalling. The glutamate analog L-glutamic acid decreased cyp19a1b mRNA and increased ccnd1 and grm6b mRNA in a dose-dependent manner. This suggests that ccnd1 and grm6b expression may be regulated by glutamate receptors other than group III mGluRs, for example, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are expressed in cultured goldfish RGCs. It was found that E2 upregulated cyp19a1b, ccnd1 and grm7 mRNA. However, selective activation of group III mGluRs decreases the stimulatory effect of E2 on ccnd1 expression. My findings show that glutamate finely regulates RGC neurogenic and steroidogenic genes, which may implicate glutamate in the regulation of RGC differentiation, RGC proliferation, and neurogenesis in surrounding cells.

abstract: Women are exposed to numerous endogenous and exogenous hormones across the lifespan. In the last several decades, the prescription of novel hormonal contraceptives and hormone therapies (HTs) have resulted in aging women that have a unique hormone exposure history; little is known about the impact of these hormone exposures on short- and long- term brain health. The goal of my dissertation was to understand how lifetime hormone exposures shape the female cognitive phenotype using several innovative approaches, including a new human spatial working memory task, the human radial arm maze (HRAM), and several rodent menopause models with variants of clinically used hormone treatments. Using the HRAM (chapter 2) and established human neuropsychological tests, I determined males outperformed females with high endogenous or exogenous estrogen levels on visuospatial tasks and the spatial working memory HRAM (chapter 3). Evaluating the synthetic estrogen in contraceptives, ethinyl estradiol (EE), I found a high EE dose impaired spatial working memory in ovariectomized (Ovx) rats, medium and high EE doses reduced choline-acetyltransferace-immunoreactive neuron population estimates in the basal forebrain following Ovx (chapter 4), and low EE impaired spatial cognition in ovary-intact rats (chapter 5). Assessing the impact of several clinically-used HTs, I identified a window of opportunity around ovarian follicular depletion outside of which the HT conjugated equine estrogens (CEE) was detrimental to spatial memory (chapter 6), as well as therapeutic potentials for synthetic contraceptive hormones (chapter 9) and bioidentical estradiol (chapter 7) during and after the transition to menopause. Chapter 6 and 7 findings, that estradiol and Ovx benefitted cognition after the menopause transition, but CEE did not, are perhaps due to the negative impact of ovarian-produced, androstenedione-derived estrone; indeed, blocking androstenedione’s conversion to estrone prevented its cognitive impairments (chapter 8). Finally, I determined that EE combined with the popular progestin levonorgestrel benefited spatial memory during the transition to menopause, a profile not seen with estradiol, levonorgestrel, or EE alone (chapter 9). This work identifies several cognitively safe, and enhancing, hormonal treatment options at different time points throughout female aging, revealing promising avenues toward optimizing female health. / Dissertation/Thesis / Doctoral Dissertation Psychology 2015

O objetivo do presente trabalho foi investigar a relação do núcleo pré-mamilar ventral (PMV): 1) na regulação do sistema reprodutivo em fêmeas, 2) como mediador dos efeitos da leptina e 3) na resposta à estimulação feromonal. Para tanto, induzimos lesão bilateral do PMV em ratas. Foi observado que a lesão do PMV promove: alteração no ciclo estral e da histologia ovariana; redução dos níveis de estradiol e LH; e supressão de vias neurais que controlam o eixo reprodutivo. Além disso, a lesão do PMV impediu que a administração central de leptina induzisse secreção de LH durante o jejum. Em outros experimentos, realizados em ratos machos, verificou-se que neurônios que sintetizam óxido nítrico no PMV e no núcleo medial da amígdala são ativados em resposta ao odor de conspecíficos, em especial o de fêmeas. Assim, foi demonstrado que o PMV exerce papel relevante na regulação do sistema reprodutivo e media a ação estimulatória da leptina na secreção do LH. Além disso, o PMV faz parte do circuito neural relacionado com a resposta aos feromônios. / The objective of this study was to assess the role played by the ventral premammillary nucleus (PMV): 1) in the regulation of the female reproductive system, 2) as a mediator of the effects of leptin and 3) in response to pheromonal stimulation. For this purpose, we produced bilateral PMV lesions in female rats. We observed that PMV lesion: disrupted the estrous cycle and altered the number of ovarian antral follicles; reduced the estradiol and LH levels; and suppressed neural pathways that control the reproductive axis. We found that lesions of the PMV blocked leptin stimulation of LH secretion during fasting. In another experiment using male rats, we showed that nitric oxide synthesizing neurons in the PMV and medial nucleus of amygdala are activated by conspecific odors, especially female odors. Thus, we showed that PMV plays an important role in the control of females reproductive system and mediates the stimulatory effects of leptin on LH secretion. Also, PMV is part of the neural circuitry related to pheromonal responses.

Diversos procedimentos são capazes de induzir a recuperação de memórias traumáticas (RecMem). Técnicas de psicoterapia têm sido usadas para reduzir a resposta emocional às memórias traumáticas, através de processos de re-estruturação e re-significação das vivências, baseados na resiliência dos sujeitos. Durante a RecMem podem ocorrer sinais clínicos de ativação autonômica que não são reações devidas a estressores externos. Pelo contrário, constituem o resultado de estímulos de natureza psíquica gerados internamente. Assim, é possível se especular se eles podem funcionar como estressores puramente psicológicos e, como tais, induzir respostas autonômicas e neuroendócrinas. O objetivo deste trabalho é o de se determinar se estas respostas ocorrem, assim como o de identificar suas características qualitativas e quantitativas. Seis voluntários de ambos os sexos, previamente selecionados e avaliados por testes psicológicos (SICD, Hamilton e BDI) e de cronotipagem, foram submetidos a duas condições: 1. condição controle (CC), e 2. condição de recuperação (CR). Em ambas as condições, os sujeitos foram colocados em ambiente controlado, com coleta de material destinado a dosagens de ACTH, adrenalina, cortisol, prolactina, TSH e GH (hormônios envolvidos com as respostas ao estresse), a cada 15 minutos, por 2 horas, com a primeira coleta 30 minutos antes do início dos procedimentos. Em ambas as condições, os sujeitos repousaram durante estes 30 minutos. Na CC os sujeitos permaneceram em repouso e na CR foram submetidos aos processos de RecMem, com seguimento psicoterapêutico subseqüente. Os dados obtidos foram avaliados quanto ao perfil das curvas tempo e resposta para cada hormônio e submetidos a testes estatísticos de comparação da secreção hormonal em ambas as condições. Concluímos que existiram respostas autonômicas e neuroendócrinas compatíveis com estresse psicológico que não são constantes em todos os sujeitos, onde se ressalta a variabilidade das respostas individuais obtidas. / Several procedures are capable to induce retrieval of traumatic memories (RecMem). Psychotherapy techniques have been used to reduce the traumatic memories emotional response through rebuilding and re-meaning of the traumatic memories based on the subject´s resilience. Clinical signs of autonomic activation may be seen during RecMem, not representing reactions to external stressors. On the contrary, they are the result of psychic inputs internally generated. Therefore, it is possible to speculate that they can act as pure psychological stressors and, as such, able to induce autonomic and neuroendocrine responses. The aim of the present report is to determine whether these responses actually happen, as well as to determine their qualitative and quantitative characteristics. Six volunteers of both sexes, previously selected, and submitted to psychological testing (SCID, Hamilton and BDI) as well as chronotype determination, were submitted to two conditions: 1. Control condition (CC) and 2. Retrieval condition (CR). The subjects were placed in a controlled environment in both conditions, with ACTH, cortisol, adrenalin, prolactin, TSH and GH (stress related hormones) blood testing at every 15 minutes, for 2 hours; the first sample was obtained 30 minutes before the beginning of the procedures. In both conditions the subjects rested during these 30 minutes. In the CC the subjects remained resting, and in the CR were submitted to RecMem procedures followed by psychotherapeutic support. The resulting data were analyzed in their time-response curve profiles as well as were submitted to hormonal secretion statistical evaluation in both conditions. We conclude for autonomic and neuroendocrine responses compatible with psychological stress, that are not the same in all subjects, high lightening the variability of individual obtained responses.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism. / Science, Faculty of / Mathematics, Department of / Graduate

Serotonin stimulates reproduction and inhibits feeding/growth in the neuroendocrine brain of goldfish. The objective of this thesis is to study the effects of selective serotonin reuptake inhibitor pharmaceuticals (SSRIs) on these systems, as SSRIs, such as fluoxetine, are detected in effluent and bioconcentrate in the brain of wild fish. Genes of the serotonin system were cloned to identify molecular conservation, seasonal expression, and tissue distribution. The serotonin transporter, the target molecule of fluoxetine, was highly conserved and ubiquitously expressed in goldfish. Seasonal changes of hypothalamic gene expression of the serotonin transporter support a role in the seasonal modulation of both processes. Fluoxetine injection experiments were used to assess effects on reproductive endpoints and to identify molecular mechanisms in the neuroendocrine brain. Fluoxetine inhibited serum estradiol concentrations in female goldfish and decreased isotocin mRNA abundance in the hypothalamus and telencephalon. Isotocin injections stimulated circulating estradiol concentrations, providing a causal link. Evidence for an involvement of serotonin in isotocin regulation was investigated using immunocytochemistry and 5-HT1A receptor agonists and antagonists. A close proximity of serotonin fibers and isotocin cell bodies and fibers was found in the telencephalon and pituitary,respectively. Injection of a 5-HT1A receptor antagonist inhibited isotocin mRNA expression in the telencephalon. Identified gene targets were investigated in waterborne fluoxetine exposures,including environmental concentrations. Waterborne fluoxetine led to a reduction in basal and pheromone-stimulated milt volume in male goldfish. Gene expression evidence indicated a central inhibitory effect of fluoxetine through the decrease in mRNA abundance of follicle-stimulating hormone in the pituitary and isotocin in the telencephalon. Feeding rate and weight decreased in fluoxetine-injected goldfish, indicating an anorexigenic effect. Fluoxetine induced changes in the gene expression of the feeding peptides neuropeptide Y, corticotropin-releasing factor, and cocaine- and amphetamine-regulated transcript-I in the hypothalamus and telencephalon. Waterborne exposure to fluoxetine validated the anorexigenic effect in goldfish and was correlated with increased expression of corticotropin-releasing factor mRNA, an anorectic peptide. The thesis provides evidence for disrupting effects of fluoxetine on neuroendocrine control of reproductive function and feeding/growth in goldfish, partially at environmental concentrations. The thesis provides the framework for the investigation of existing aquatic contaminants which modulate the serotonin system.