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Periventricular nucleus kisspeptin neurons project to the perinuclear zone of the supraoptic nucleus. A Seymour, R Campbell, C Brown. Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.Oxytocin is a hormone synthesised in supraoptic nucleus (SON) neurons and is important for parturition. However, the mechanisms that regulate oxytocin neuronal firing during parturition are poorly understood. We have shown that intracerebroventricular kisspeptin increases oxytocin neuron firing rate in late pregnant rats, but not virgin rats in vivo. Furthermore, there is an increase in kisspeptin fibre density in the perinuclear zone (PNZ), immediately dorsal to the SON. We aim to investigate a potential role for kisspeptin to regulate oxytocin release in late pregnancy to prepare for parturition.Here we have used retrograde tracing and immunohistochemistry to study the origin of PNZ kisspeptin fibres in virgin and late pregnant (day 21) rats. Under isofluorane anaesthesia, green fluorescent microspheres were stereotaxically injected into the PNZ. Following 4 -7 days recovery, rats were anaesthetised with 60 mg/kg pentobarbitone and perfused. Brains were sliced and labelled for kisspeptin. The average numbers of cells per section expressing kisspeptin, tracer or both were counted and compared between groups.Only kisspeptin-positive cells in the periventricular nucleus (PeN) co-expressed tracer. There was a significant increase in the number of cells expressing kisspeptin in late pregnant rats (7.7 ± 1.7 mean ± SEM, n = 4) compared to virgin rats (2.1 ± 0.6, n = 6, P = 0.007, student's t test). There was also a significant increase in the number of cells co-expressing kisspeptin and tracer in late pregnant rats (1.4 ± 0.26,) compared to virgin rats (0.43 ± 0.27, P = 0.034). However, the number of tracer-positive cells was similar in virgin (18.4 ± 3.9) and late pregnant rats (16.6 ± 2.6, P = 0.74).These results show that PeN kisspeptin neurons project to the PNZ. Hence, these neurons might act as a positive regulator for oxytocin release at parturition to aid delivery of the offspring. Enhanced sympathetic activity contributing to seizure-induced cardiomyopathy: Therapeutic option?M Read, D McCann, R Millen, J Harrison, S Kerr, I Sammut. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.Seizures are associated with enhanced sympathetic activity resulting in cardiac dysfunction and structural damage. The current study examined ictal electrocardiographic (ECG) activity up to 7 days following seizure induction. It was hypothesised that intervention with atenolol, a peripheral β1 antagonist, could attenuate this pathology.Male Sprague-Dawley rats (320-350g) were implanted with ECG electrodes to allow telemetric recordings during seizures. Baseline 30 min recordings were taken prior to seizure-induction by intrahippocampal kainic acid (2 nmol, 1 µl/min). Animals were treated with saline or atenolol (5 mg/kg) 60 min post-seizure induction (n = 12/group) and for the remainder of the study (up to 14 days). Control animals were administered intrahippocampal saline and subcutaneous saline at 60 min. Data was analysed using a 2-way repeated measures ANOVA.Seizures were associated with a significant increases in heart rate (HR, 36 ± 2.7%) and blood pressure (20 ± 6.5%) above baseline (P < 0.05) suggesting elevated sympathetic activity. Troponin I levels were elevated 24 hours (0.61 ± 0.03 ng/ml) following seizure induction (0.15 ± 0.02 ng/ml at baseline, P < 0.05), demonstrating acute myocardial damage. Echocardiography at 7 and 14 days revealed deterioration in cardiac function as determined by decreased ejection fractional (80 ± 5% and 75 ± 1%, respectively). Histological examination in the saline animals indicated the presence of reversible ischaemic damage, fibrosis and inflammatory cell filtration, contributing to increased susceptibility to aconitine-induced arrhythmias. Atenolol intervention was effective at reducing the HR, QTc interval and systolic blood pressure back to baseline. Furthermore, atenolol treatment preserved cardiac function and prevented structural damage at 7 and 14 days, reducing the risk of arrhythmia.These results show that seizures are associated with enhanced sympathetic modulation resulting in altered ECG activity and cardiac pathology. Atenolol intervention provides a promising therapeutic approach to seizure-induced cardiomyopathy. Colorectal tumour associated macrophages are more pro-inflammatory than adjacent control bowel tissue macrophage populations. S Norton1, E Taylor1, E Dunn1, F Munro2, M Black3, J McCall2, R Kemp1. 1Department of Microbiology and Immunology, Otago School of Medical Sciences, 2Department of Surgical Sciences, Dunedin School of Medicine, 3Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin.A high infiltration of macrophages in colorectal cancer is associated with improved prognosis for patients, the opposite to what is observed in many other cancers. The objective of this project was to characterise colorectal tumour macrophages to help explain this association.Human macrophage functional phenotypes were defined in vitro using multicolour flow cytometry, ELISA and qPCR. Corresponding populations were subsequently detected, using flow cytometry, in both tumour tissue and non-transformed bowel tissue from colorectal cancer patients.A significant greater frequency of inflammatory macrophages (CD45+CD33-CD14lo/-CD11b+CD64+CD206int) (tumour = 0.99 ± 0.26, control bowel = 0.27 ± 0.11, mean % total live immune cells ± SEM, Wilcoxon matched-pairs signed rank test, P = 0.02, n = 7) was observed in the tumour tissue of each patient. Analysis of patient samples revealed other myeloid derived immune cells. The frequency of myeloid derived suppressor cells (CD45+CD33+CD11b+CD64-CD14-) was significantly increased (tumour = 0.64 ± 0.22, control bowel = 0.26 ± 0.07, P = 0.03, n = 7) in tumour tissue compared to control bowel. A large population of gut resident macrophages was observed in both tumour and control bowel tissue. These macrophages are highly phagocytic and unresponsive to cytokines – a phenotype that could be beneficial in restricting tumour growth. Here, this population could be subdivided into gut resident (CD45+CD33+CD14-CD11b-CD64-) and inflammatory gut resident (CD45+CD33+CD14+CD11b+CD64+) macrophages. Macrophages (CD45+CD64+CD11b+), sorted from both tumour and control bowel tissue, were cultured with T cells (CD3+) sorted from patient blood. Tumour macrophages increased T cell IFN-g and IL-17 production, as measured by flow cytometry, compared to T cell cultured alone, whereas control bowel macrophages did not.This study highlights the complexity of macrophages and provides methods to examine them ex vivo from human tissue. Data gained from this study could be used for future investigation of macrophages as both prognostic and therapeutic targets. Optogenetic activation of gonadotropin-releasing hormone neurons reveals minimal requirements for pulsatile luteinising hormone secretion. P Campos, A Herbison, B Hyland. Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.The reproductive system is critically dependent upon patterned pulsatile hormone release that is driven by the gonadotropin-releasing hormone (GnRH) neurons. The patterns of neuronal activity responsible for generating the pulsatile release of GnRH are unknown. We developed a methodology in mice for remotely controlling the activity of the GnRH neurons in vivo to establish the parameters of activation required to evoke a pulse of luteinising hormone (LH) secretion from the pituitary.Injections of Cre-dependent channelrhodopsin (ChR2)-bearing adeno-associated virus into the median eminence of GnRH-Cre mice resulted in the selective expression of ChR2 in GnRH neurons (93 ± 8 % double-labeled neurons, mean ± sem, n = 6, in the rostral preoptic area (rPOA)). Acute brain slice experiments demonstrated that ChR2-expressing GnRH neurons could be driven to fire with high spike fidelity with blue light stimulation frequencies up to 40 Hz. In vivo, optical fibers were implanted in the vicinity of GnRH neurons within the rPOA of anesthetised, ovariectomised mice. Optogenetic activation of GnRH neurons for 30 s to five min time periods over a range of different frequencies revealed that 10 Hz stimulation for two minutes was the minimum required to generate a pulse-like increment of LH (1.49 ± 0.08 fold increase, n = 8) which was significantly different (P < 0.001, one-way ANOVA) from control animals. Under these conditions, the dynamics of optogenetically-evoked LH secretion are very similar to that of endogenous LH pulses with the rise time and increment in LH being identical. This suggests that the minimal parameters of GnRH neuron activation reported here are likely to be that occurring in vivo for pulsatile LH secretion.This study provides the first major insight into the nature of GnRH neuron activation required to generate pulsatile LH secretion and provides the foundations for understanding pulsatility within the reproductive axis. Triazole drug target structures in a resistant yeast strain. A Sagatova1, M Keniya1, F Huschmann1,2, S Willbanks3, R Cannon1, J Tyndall2, B Monk1. 1Sir John Walsh Research Institute and Department of Oral Sciences, Faculty of Dentistry, 2School of Pharmacy, 3Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin.Fungal pathogens are estimated to be responsible for ~1.5 million deaths annually. Triazole drugs provide some of the most widely used treatments of fungal infections. They target the membrane protein lanosterol 14α-demethylase (Erg11p) which catalyses the rate limiting step of ergosterol biosynthesis. Inhibition of Erg11p depletes cell membranes of ergosterol and inhibits fungal growth. Resistance to triazoles can arise due to mutations in Erg11p that reduce binding affinity of the drug. An increasing incidence of resistance in fungal pathogens such as Candida species is of major concern. This project aims to understand how a mutation in Erg11p differentially affects the binding of triazoles.Wild type hexahistidine-tagged ERG11 or a mutant allele that translates to a single amino acid change found in clinical isolates of pathogenic fungi was cloned in a Saccharomyces cerevisiae membrane protein overexpression system. Endogenous ERG11 was deleted. Recombinant ScErg11p6×His was solubilised with N-decyl-β-D-maltoside and purified by Ni-NTA affinity chromatography and size exclusion chromatography. Purified enzyme was used for ligand binding assays and crystallographic studies. X-ray crystal structures of the ScErg11p Y140F mutant in complex with itraconazole (ITC) and fluconazole (FLC) were determined at a resolution of 2 Å from crystallographic data collected at the Australian Synchrotron.The Candida albicans Erg11p Y132F mutation gives resistance to FLC in clinical isolates. The equivalent Y140F mutation in ScErg11p conferred resistance to FLC but not ITC. The ScErg11p Y140F structure with the long-tailed triazole ITC revealed binding matching that of the wild-type, with an additional water molecule between a heme carboxyl and the aromatic side chain of F140. Comparison of wild type ScErg11p structure to the Y140F mutant in complex with FLC revealed a disruption of well-ordered hydrogen bonding interactions between the enzyme and the drug. Disruption of this hydrogen bonding network helps to explain FLC resistance of the mutant enzyme. Modulation of heart rate by specific β1- and β2-adrenoceptor stimulation in conscious Zucker type II Diabetic Fatty rats. R Cook, C Bussey, P Cragg, R Lamberts. Department of Physiology and HeartOtago, Otago School of Medical Sciences, University of Otago, Dunedin.Resting heart rate (HR) is the strongest predictor of mortality in patients with cardiac disease. The β-adrenoceptor (β-AR) functional responsiveness and β1-AR expression, key components of sinoatrial rate regulation, are reduced in many cardiomyopathies. An in vitro model suggests co-localisation of ‘funny'-channels and β2-ARs via lipid-rich caveolae is the mechanism through which HR is modulated. β2-AR redistribution in the failing heart compensates for the decreased β1-AR function. Whether similar β2-AR subtype changes contribute to HR dysfunction in diabetes is unknown. We aimed to determine the effects of specific β1- and β2-AR stimulation on HR in type II diabetes in vivo.Male Zucker Diabetic Fatty (ZDF) rats and lean littermates (20-week old, n = 8) were implanted with a radiotelemeter to measure arterial blood pressure and derive HR and a vascular access port to inject drugs intravenously. Cumulative doses of isoproterenol (non-selective β1- and β2-AR agonist, 0.001 – 3 µg.kg-1) or fenoterol (non-selective β2-AR agonist, 0.001 – 3 µg.kg-1) in the presence or absence of atenolol (selective β1-AR antagonist, 2000 µg.kg-1) were injected.Increases in HR due to non-selective β-AR stimulation were not significantly different between groups across doses (at 0.1 µg.kg-1: 106 ± 10 vs. 109 ± 13 bpm). However the HR response to specific β2-AR stimulation was significantly reduced in ZDF rats (25 ± 4 vs. 2 ± 4 bpm and 47 ± 7 vs. 20 ± 2 bpm; P < 0.002 at 0.03 and 0.1 µg.kg-1, respectively). HR responses adjusted to the baseline were modulated by β2-AR in controls although in ZDF rats HR regulation involved both β1- and β2-ARs.Thus, in conscious ZDF rats β-AR responsiveness was not affected, whereas the role of β1-ARs was increased. Therefore, HR regulation by β1- and β2-AR is altered in diabetes, and β1-AR signaling might play a compensatory role in maintaining β-AR reactivity. Polymerase-1 and transcript release factor and caveolae in prostate cancer; which is more important? J-Y Low, H Nicholson. Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.Prostate cancer poses a significant health threat to men world-wide. Caveolae are “cave-like” invaginations that are found in the plasma membrane. Polymerase-1 and transcript release factor (PTRF) is reported to be involved in the formation of caveolae. Expression of PTRF and caveolae are lost in prostate cancer cell lines and tissues while restoration of PTRF expression leads to formation of caveolae and reduced aggressive phenotypes in vitro and in vivo. However it is unclear whether the effect seen is due to PTRF itself or the restoration of caveolae. This study examines whether PTRF or caveolae are involved in prostate cancer progression.Using normal prostate epithelial cells, RWPE-1, PTRF was knocked down with 120 nM siRNA, with scrambled siRNA used as control, and caveolae were disrupted with 10 mM of methyl-β-cyclodextrin (MBCD). Transmission electron microscopy was performed to determine the number of caveolae. Cell proliferation was measured with CellTiter 96 aqueous non-radioactive cell proliferation kit. Migration and invasion assays were performed with Transwell migration and Matrigel invasion inserts respectively. Changes in cellular signalling were examined with Pathscan intracellular signalling array. Statistical analyses were computed with Student's unpaired t-test.Knock down PTRF, or MBCD treatment, reduced caveolae numbers in RWPE-1 cells (P < 0.05 siRNA; P < 0.01 MBCD, n = 50 cells). Following both treatments increased proliferation (P < 0.05, n = 6), migration (P < 0.05, n = 3) and invasion (P < 0.001 siRNA; P < 0.01 MBCD, n = 3) was observed. However, cell-signalling arrays showed a different pattern of signaling molecules activation. Two fold increase in p-STAT3 (P < 0.05, n = 4) was seen after knocking down PTRF, while disrupting caveolae resulted in two fold increase of p-ERK1/2 (P < 0.01, n = 4).Both PTRF and caveolae may be involved in prostate cancer progression, however these elements may drive prostate cancer progression through different signaling pathways. Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome. A Moore, M Prescott, R Campbell. Department of Physiology and Centre for Neuroendocrinology, Otago School of Medical Sciences, University of Otago, Dunedin.Polycystic ovarian syndrome (PCOS) is the most common cause of infertility among women of reproductive age. An increase in luteinising hormone (LH) pulse frequency associated with PCOS is suggestive of changes in gonadotropin-releasing hormone (GnRH) neuron regulation. We hypothesise that altered neuronal regulation of GnRH neurons is due to changes in their afferent synaptic input.PCOS was modeled in mice by prenatal androgen (PNA) exposure. GnRH-green fluorescent protein (GFP) mice were used to assess synaptic input to GnRH neurons in control and PNA-treated mice (n = 8 / group). Immunohistochemical labeling of brain sections revealed a significant increase in the density of vesicular GABA transporter (vGaT) appositions with GnRH neurons in PNA-treated mice (0.8 ± 0.08 appositions/μm) compared to controls (0.4 ± 0.02 appositions/μm, P < 0.05, two-tailed t-test). As GABA signaling to GnRH neurons can be excitatory, increased GABAergic input may elevate GnRH neuron activity. However, the identity of GABAergic neuronal populations regulating GnRH neuron activity remains largely unknown.To address whether increased GABA innervation of GnRH neurons originates in the arcuate nucleus (ARN), a Cre-dependent adenoviral vector expressing farnesylated enhanced GFP (EGFPf) was injected into the ARN of vGaT-Cre mice. Immunofluorescent labeling revealed that EGFPf-positive ARN GABAergic projections heavily contacted and even bundled with GnRH neuron dendrites. In control and PNA-treated mice, 61 ± 6.1 % and 60 ± 10.5 % of GnRH neurons closely apposed projections from ARN GABAergic neurons, respectively (n = 6 / group). Furthermore, the density of fibres apposing the GnRH neuron dendrite was even greater in PNA-treated mice (0.09 ± 0.02 apposing fibres/μm) compared to controls (0.04 ± 0.006 apposing fibres/μm, P < 0.05).These data describe a novel, robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome. \r\n

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Periventricular nucleus kisspeptin neurons project to the perinuclear zone of the supraoptic nucleus. A Seymour, R Campbell, C Brown. Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.Oxytocin is a hormone synthesised in supraoptic nucleus (SON) neurons and is important for parturition. However, the mechanisms that regulate oxytocin neuronal firing during parturition are poorly understood. We have shown that intracerebroventricular kisspeptin increases oxytocin neuron firing rate in late pregnant rats, but not virgin rats in vivo. Furthermore, there is an increase in kisspeptin fibre density in the perinuclear zone (PNZ), immediately dorsal to the SON. We aim to investigate a potential role for kisspeptin to regulate oxytocin release in late pregnancy to prepare for parturition.Here we have used retrograde tracing and immunohistochemistry to study the origin of PNZ kisspeptin fibres in virgin and late pregnant (day 21) rats. Under isofluorane anaesthesia, green fluorescent microspheres were stereotaxically injected into the PNZ. Following 4 -7 days recovery, rats were anaesthetised with 60 mg/kg pentobarbitone and perfused. Brains were sliced and labelled for kisspeptin. The average numbers of cells per section expressing kisspeptin, tracer or both were counted and compared between groups.Only kisspeptin-positive cells in the periventricular nucleus (PeN) co-expressed tracer. There was a significant increase in the number of cells expressing kisspeptin in late pregnant rats (7.7 ± 1.7 mean ± SEM, n = 4) compared to virgin rats (2.1 ± 0.6, n = 6, P = 0.007, student's t test). There was also a significant increase in the number of cells co-expressing kisspeptin and tracer in late pregnant rats (1.4 ± 0.26,) compared to virgin rats (0.43 ± 0.27, P = 0.034). However, the number of tracer-positive cells was similar in virgin (18.4 ± 3.9) and late pregnant rats (16.6 ± 2.6, P = 0.74).These results show that PeN kisspeptin neurons project to the PNZ. Hence, these neurons might act as a positive regulator for oxytocin release at parturition to aid delivery of the offspring. Enhanced sympathetic activity contributing to seizure-induced cardiomyopathy: Therapeutic option?M Read, D McCann, R Millen, J Harrison, S Kerr, I Sammut. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.Seizures are associated with enhanced sympathetic activity resulting in cardiac dysfunction and structural damage. The current study examined ictal electrocardiographic (ECG) activity up to 7 days following seizure induction. It was hypothesised that intervention with atenolol, a peripheral β1 antagonist, could attenuate this pathology.Male Sprague-Dawley rats (320-350g) were implanted with ECG electrodes to allow telemetric recordings during seizures. Baseline 30 min recordings were taken prior to seizure-induction by intrahippocampal kainic acid (2 nmol, 1 µl/min). Animals were treated with saline or atenolol (5 mg/kg) 60 min post-seizure induction (n = 12/group) and for the remainder of the study (up to 14 days). Control animals were administered intrahippocampal saline and subcutaneous saline at 60 min. Data was analysed using a 2-way repeated measures ANOVA.Seizures were associated with a significant increases in heart rate (HR, 36 ± 2.7%) and blood pressure (20 ± 6.5%) above baseline (P < 0.05) suggesting elevated sympathetic activity. Troponin I levels were elevated 24 hours (0.61 ± 0.03 ng/ml) following seizure induction (0.15 ± 0.02 ng/ml at baseline, P < 0.05), demonstrating acute myocardial damage. Echocardiography at 7 and 14 days revealed deterioration in cardiac function as determined by decreased ejection fractional (80 ± 5% and 75 ± 1%, respectively). Histological examination in the saline animals indicated the presence of reversible ischaemic damage, fibrosis and inflammatory cell filtration, contributing to increased susceptibility to aconitine-induced arrhythmias. Atenolol intervention was effective at reducing the HR, QTc interval and systolic blood pressure back to baseline. Furthermore, atenolol treatment preserved cardiac function and prevented structural damage at 7 and 14 days, reducing the risk of arrhythmia.These results show that seizures are associated with enhanced sympathetic modulation resulting in altered ECG activity and cardiac pathology. Atenolol intervention provides a promising therapeutic approach to seizure-induced cardiomyopathy. Colorectal tumour associated macrophages are more pro-inflammatory than adjacent control bowel tissue macrophage populations. S Norton1, E Taylor1, E Dunn1, F Munro2, M Black3, J McCall2, R Kemp1. 1Department of Microbiology and Immunology, Otago School of Medical Sciences, 2Department of Surgical Sciences, Dunedin School of Medicine, 3Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin.A high infiltration of macrophages in colorectal cancer is associated with improved prognosis for patients, the opposite to what is observed in many other cancers. The objective of this project was to characterise colorectal tumour macrophages to help explain this association.Human macrophage functional phenotypes were defined in vitro using multicolour flow cytometry, ELISA and qPCR. Corresponding populations were subsequently detected, using flow cytometry, in both tumour tissue and non-transformed bowel tissue from colorectal cancer patients.A significant greater frequency of inflammatory macrophages (CD45+CD33-CD14lo/-CD11b+CD64+CD206int) (tumour = 0.99 ± 0.26, control bowel = 0.27 ± 0.11, mean % total live immune cells ± SEM, Wilcoxon matched-pairs signed rank test, P = 0.02, n = 7) was observed in the tumour tissue of each patient. Analysis of patient samples revealed other myeloid derived immune cells. The frequency of myeloid derived suppressor cells (CD45+CD33+CD11b+CD64-CD14-) was significantly increased (tumour = 0.64 ± 0.22, control bowel = 0.26 ± 0.07, P = 0.03, n = 7) in tumour tissue compared to control bowel. A large population of gut resident macrophages was observed in both tumour and control bowel tissue. These macrophages are highly phagocytic and unresponsive to cytokines – a phenotype that could be beneficial in restricting tumour growth. Here, this population could be subdivided into gut resident (CD45+CD33+CD14-CD11b-CD64-) and inflammatory gut resident (CD45+CD33+CD14+CD11b+CD64+) macrophages. Macrophages (CD45+CD64+CD11b+), sorted from both tumour and control bowel tissue, were cultured with T cells (CD3+) sorted from patient blood. Tumour macrophages increased T cell IFN-g and IL-17 production, as measured by flow cytometry, compared to T cell cultured alone, whereas control bowel macrophages did not.This study highlights the complexity of macrophages and provides methods to examine them ex vivo from human tissue. Data gained from this study could be used for future investigation of macrophages as both prognostic and therapeutic targets. Optogenetic activation of gonadotropin-releasing hormone neurons reveals minimal requirements for pulsatile luteinising hormone secretion. P Campos, A Herbison, B Hyland. Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.The reproductive system is critically dependent upon patterned pulsatile hormone release that is driven by the gonadotropin-releasing hormone (GnRH) neurons. The patterns of neuronal activity responsible for generating the pulsatile release of GnRH are unknown. We developed a methodology in mice for remotely controlling the activity of the GnRH neurons in vivo to establish the parameters of activation required to evoke a pulse of luteinising hormone (LH) secretion from the pituitary.Injections of Cre-dependent channelrhodopsin (ChR2)-bearing adeno-associated virus into the median eminence of GnRH-Cre mice resulted in the selective expression of ChR2 in GnRH neurons (93 ± 8 % double-labeled neurons, mean ± sem, n = 6, in the rostral preoptic area (rPOA)). Acute brain slice experiments demonstrated that ChR2-expressing GnRH neurons could be driven to fire with high spike fidelity with blue light stimulation frequencies up to 40 Hz. In vivo, optical fibers were implanted in the vicinity of GnRH neurons within the rPOA of anesthetised, ovariectomised mice. Optogenetic activation of GnRH neurons for 30 s to five min time periods over a range of different frequencies revealed that 10 Hz stimulation for two minutes was the minimum required to generate a pulse-like increment of LH (1.49 ± 0.08 fold increase, n = 8) which was significantly different (P < 0.001, one-way ANOVA) from control animals. Under these conditions, the dynamics of optogenetically-evoked LH secretion are very similar to that of endogenous LH pulses with the rise time and increment in LH being identical. This suggests that the minimal parameters of GnRH neuron activation reported here are likely to be that occurring in vivo for pulsatile LH secretion.This study provides the first major insight into the nature of GnRH neuron activation required to generate pulsatile LH secretion and provides the foundations for understanding pulsatility within the reproductive axis. Triazole drug target structures in a resistant yeast strain. A Sagatova1, M Keniya1, F Huschmann1,2, S Willbanks3, R Cannon1, J Tyndall2, B Monk1. 1Sir John Walsh Research Institute and Department of Oral Sciences, Faculty of Dentistry, 2School of Pharmacy, 3Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin.Fungal pathogens are estimated to be responsible for ~1.5 million deaths annually. Triazole drugs provide some of the most widely used treatments of fungal infections. They target the membrane protein lanosterol 14α-demethylase (Erg11p) which catalyses the rate limiting step of ergosterol biosynthesis. Inhibition of Erg11p depletes cell membranes of ergosterol and inhibits fungal growth. Resistance to triazoles can arise due to mutations in Erg11p that reduce binding affinity of the drug. An increasing incidence of resistance in fungal pathogens such as Candida species is of major concern. This project aims to understand how a mutation in Erg11p differentially affects the binding of triazoles.Wild type hexahistidine-tagged ERG11 or a mutant allele that translates to a single amino acid change found in clinical isolates of pathogenic fungi was cloned in a Saccharomyces cerevisiae membrane protein overexpression system. Endogenous ERG11 was deleted. Recombinant ScErg11p6×His was solubilised with N-decyl-β-D-maltoside and purified by Ni-NTA affinity chromatography and size exclusion chromatography. Purified enzyme was used for ligand binding assays and crystallographic studies. X-ray crystal structures of the ScErg11p Y140F mutant in complex with itraconazole (ITC) and fluconazole (FLC) were determined at a resolution of 2 Å from crystallographic data collected at the Australian Synchrotron.The Candida albicans Erg11p Y132F mutation gives resistance to FLC in clinical isolates. The equivalent Y140F mutation in ScErg11p conferred resistance to FLC but not ITC. The ScErg11p Y140F structure with the long-tailed triazole ITC revealed binding matching that of the wild-type, with an additional water molecule between a heme carboxyl and the aromatic side chain of F140. Comparison of wild type ScErg11p structure to the Y140F mutant in complex with FLC revealed a disruption of well-ordered hydrogen bonding interactions between the enzyme and the drug. Disruption of this hydrogen bonding network helps to explain FLC resistance of the mutant enzyme. Modulation of heart rate by specific β1- and β2-adrenoceptor stimulation in conscious Zucker type II Diabetic Fatty rats. R Cook, C Bussey, P Cragg, R Lamberts. Department of Physiology and HeartOtago, Otago School of Medical Sciences, University of Otago, Dunedin.Resting heart rate (HR) is the strongest predictor of mortality in patients with cardiac disease. The β-adrenoceptor (β-AR) functional responsiveness and β1-AR expression, key components of sinoatrial rate regulation, are reduced in many cardiomyopathies. An in vitro model suggests co-localisation of ‘funny'-channels and β2-ARs via lipid-rich caveolae is the mechanism through which HR is modulated. β2-AR redistribution in the failing heart compensates for the decreased β1-AR function. Whether similar β2-AR subtype changes contribute to HR dysfunction in diabetes is unknown. We aimed to determine the effects of specific β1- and β2-AR stimulation on HR in type II diabetes in vivo.Male Zucker Diabetic Fatty (ZDF) rats and lean littermates (20-week old, n = 8) were implanted with a radiotelemeter to measure arterial blood pressure and derive HR and a vascular access port to inject drugs intravenously. Cumulative doses of isoproterenol (non-selective β1- and β2-AR agonist, 0.001 – 3 µg.kg-1) or fenoterol (non-selective β2-AR agonist, 0.001 – 3 µg.kg-1) in the presence or absence of atenolol (selective β1-AR antagonist, 2000 µg.kg-1) were injected.Increases in HR due to non-selective β-AR stimulation were not significantly different between groups across doses (at 0.1 µg.kg-1: 106 ± 10 vs. 109 ± 13 bpm). However the HR response to specific β2-AR stimulation was significantly reduced in ZDF rats (25 ± 4 vs. 2 ± 4 bpm and 47 ± 7 vs. 20 ± 2 bpm; P < 0.002 at 0.03 and 0.1 µg.kg-1, respectively). HR responses adjusted to the baseline were modulated by β2-AR in controls although in ZDF rats HR regulation involved both β1- and β2-ARs.Thus, in conscious ZDF rats β-AR responsiveness was not affected, whereas the role of β1-ARs was increased. Therefore, HR regulation by β1- and β2-AR is altered in diabetes, and β1-AR signaling might play a compensatory role in maintaining β-AR reactivity. Polymerase-1 and transcript release factor and caveolae in prostate cancer; which is more important? J-Y Low, H Nicholson. Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.Prostate cancer poses a significant health threat to men world-wide. Caveolae are “cave-like” invaginations that are found in the plasma membrane. Polymerase-1 and transcript release factor (PTRF) is reported to be involved in the formation of caveolae. Expression of PTRF and caveolae are lost in prostate cancer cell lines and tissues while restoration of PTRF expression leads to formation of caveolae and reduced aggressive phenotypes in vitro and in vivo. However it is unclear whether the effect seen is due to PTRF itself or the restoration of caveolae. This study examines whether PTRF or caveolae are involved in prostate cancer progression.Using normal prostate epithelial cells, RWPE-1, PTRF was knocked down with 120 nM siRNA, with scrambled siRNA used as control, and caveolae were disrupted with 10 mM of methyl-β-cyclodextrin (MBCD). Transmission electron microscopy was performed to determine the number of caveolae. Cell proliferation was measured with CellTiter 96 aqueous non-radioactive cell proliferation kit. Migration and invasion assays were performed with Transwell migration and Matrigel invasion inserts respectively. Changes in cellular signalling were examined with Pathscan intracellular signalling array. Statistical analyses were computed with Student's unpaired t-test.Knock down PTRF, or MBCD treatment, reduced caveolae numbers in RWPE-1 cells (P < 0.05 siRNA; P < 0.01 MBCD, n = 50 cells). Following both treatments increased proliferation (P < 0.05, n = 6), migration (P < 0.05, n = 3) and invasion (P < 0.001 siRNA; P < 0.01 MBCD, n = 3) was observed. However, cell-signalling arrays showed a different pattern of signaling molecules activation. Two fold increase in p-STAT3 (P < 0.05, n = 4) was seen after knocking down PTRF, while disrupting caveolae resulted in two fold increase of p-ERK1/2 (P < 0.01, n = 4).Both PTRF and caveolae may be involved in prostate cancer progression, however these elements may drive prostate cancer progression through different signaling pathways. Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome. A Moore, M Prescott, R Campbell. Department of Physiology and Centre for Neuroendocrinology, Otago School of Medical Sciences, University of Otago, Dunedin.Polycystic ovarian syndrome (PCOS) is the most common cause of infertility among women of reproductive age. An increase in luteinising hormone (LH) pulse frequency associated with PCOS is suggestive of changes in gonadotropin-releasing hormone (GnRH) neuron regulation. We hypothesise that altered neuronal regulation of GnRH neurons is due to changes in their afferent synaptic input.PCOS was modeled in mice by prenatal androgen (PNA) exposure. GnRH-green fluorescent protein (GFP) mice were used to assess synaptic input to GnRH neurons in control and PNA-treated mice (n = 8 / group). Immunohistochemical labeling of brain sections revealed a significant increase in the density of vesicular GABA transporter (vGaT) appositions with GnRH neurons in PNA-treated mice (0.8 ± 0.08 appositions/μm) compared to controls (0.4 ± 0.02 appositions/μm, P < 0.05, two-tailed t-test). As GABA signaling to GnRH neurons can be excitatory, increased GABAergic input may elevate GnRH neuron activity. However, the identity of GABAergic neuronal populations regulating GnRH neuron activity remains largely unknown.To address whether increased GABA innervation of GnRH neurons originates in the arcuate nucleus (ARN), a Cre-dependent adenoviral vector expressing farnesylated enhanced GFP (EGFPf) was injected into the ARN of vGaT-Cre mice. Immunofluorescent labeling revealed that EGFPf-positive ARN GABAergic projections heavily contacted and even bundled with GnRH neuron dendrites. In control and PNA-treated mice, 61 ± 6.1 % and 60 ± 10.5 % of GnRH neurons closely apposed projections from ARN GABAergic neurons, respectively (n = 6 / group). Furthermore, the density of fibres apposing the GnRH neuron dendrite was even greater in PNA-treated mice (0.09 ± 0.02 apposing fibres/μm) compared to controls (0.04 ± 0.006 apposing fibres/μm, P < 0.05).These data describe a novel, robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome. \r\n

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Periventricular nucleus kisspeptin neurons project to the perinuclear zone of the supraoptic nucleus. A Seymour, R Campbell, C Brown. Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.Oxytocin is a hormone synthesised in supraoptic nucleus (SON) neurons and is important for parturition. However, the mechanisms that regulate oxytocin neuronal firing during parturition are poorly understood. We have shown that intracerebroventricular kisspeptin increases oxytocin neuron firing rate in late pregnant rats, but not virgin rats in vivo. Furthermore, there is an increase in kisspeptin fibre density in the perinuclear zone (PNZ), immediately dorsal to the SON. We aim to investigate a potential role for kisspeptin to regulate oxytocin release in late pregnancy to prepare for parturition.Here we have used retrograde tracing and immunohistochemistry to study the origin of PNZ kisspeptin fibres in virgin and late pregnant (day 21) rats. Under isofluorane anaesthesia, green fluorescent microspheres were stereotaxically injected into the PNZ. Following 4 -7 days recovery, rats were anaesthetised with 60 mg/kg pentobarbitone and perfused. Brains were sliced and labelled for kisspeptin. The average numbers of cells per section expressing kisspeptin, tracer or both were counted and compared between groups.Only kisspeptin-positive cells in the periventricular nucleus (PeN) co-expressed tracer. There was a significant increase in the number of cells expressing kisspeptin in late pregnant rats (7.7 ± 1.7 mean ± SEM, n = 4) compared to virgin rats (2.1 ± 0.6, n = 6, P = 0.007, student's t test). There was also a significant increase in the number of cells co-expressing kisspeptin and tracer in late pregnant rats (1.4 ± 0.26,) compared to virgin rats (0.43 ± 0.27, P = 0.034). However, the number of tracer-positive cells was similar in virgin (18.4 ± 3.9) and late pregnant rats (16.6 ± 2.6, P = 0.74).These results show that PeN kisspeptin neurons project to the PNZ. Hence, these neurons might act as a positive regulator for oxytocin release at parturition to aid delivery of the offspring. Enhanced sympathetic activity contributing to seizure-induced cardiomyopathy: Therapeutic option?M Read, D McCann, R Millen, J Harrison, S Kerr, I Sammut. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.Seizures are associated with enhanced sympathetic activity resulting in cardiac dysfunction and structural damage. The current study examined ictal electrocardiographic (ECG) activity up to 7 days following seizure induction. It was hypothesised that intervention with atenolol, a peripheral β1 antagonist, could attenuate this pathology.Male Sprague-Dawley rats (320-350g) were implanted with ECG electrodes to allow telemetric recordings during seizures. Baseline 30 min recordings were taken prior to seizure-induction by intrahippocampal kainic acid (2 nmol, 1 µl/min). Animals were treated with saline or atenolol (5 mg/kg) 60 min post-seizure induction (n = 12/group) and for the remainder of the study (up to 14 days). Control animals were administered intrahippocampal saline and subcutaneous saline at 60 min. Data was analysed using a 2-way repeated measures ANOVA.Seizures were associated with a significant increases in heart rate (HR, 36 ± 2.7%) and blood pressure (20 ± 6.5%) above baseline (P < 0.05) suggesting elevated sympathetic activity. Troponin I levels were elevated 24 hours (0.61 ± 0.03 ng/ml) following seizure induction (0.15 ± 0.02 ng/ml at baseline, P < 0.05), demonstrating acute myocardial damage. Echocardiography at 7 and 14 days revealed deterioration in cardiac function as determined by decreased ejection fractional (80 ± 5% and 75 ± 1%, respectively). Histological examination in the saline animals indicated the presence of reversible ischaemic damage, fibrosis and inflammatory cell filtration, contributing to increased susceptibility to aconitine-induced arrhythmias. Atenolol intervention was effective at reducing the HR, QTc interval and systolic blood pressure back to baseline. Furthermore, atenolol treatment preserved cardiac function and prevented structural damage at 7 and 14 days, reducing the risk of arrhythmia.These results show that seizures are associated with enhanced sympathetic modulation resulting in altered ECG activity and cardiac pathology. Atenolol intervention provides a promising therapeutic approach to seizure-induced cardiomyopathy. Colorectal tumour associated macrophages are more pro-inflammatory than adjacent control bowel tissue macrophage populations. S Norton1, E Taylor1, E Dunn1, F Munro2, M Black3, J McCall2, R Kemp1. 1Department of Microbiology and Immunology, Otago School of Medical Sciences, 2Department of Surgical Sciences, Dunedin School of Medicine, 3Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin.A high infiltration of macrophages in colorectal cancer is associated with improved prognosis for patients, the opposite to what is observed in many other cancers. The objective of this project was to characterise colorectal tumour macrophages to help explain this association.Human macrophage functional phenotypes were defined in vitro using multicolour flow cytometry, ELISA and qPCR. Corresponding populations were subsequently detected, using flow cytometry, in both tumour tissue and non-transformed bowel tissue from colorectal cancer patients.A significant greater frequency of inflammatory macrophages (CD45+CD33-CD14lo/-CD11b+CD64+CD206int) (tumour = 0.99 ± 0.26, control bowel = 0.27 ± 0.11, mean % total live immune cells ± SEM, Wilcoxon matched-pairs signed rank test, P = 0.02, n = 7) was observed in the tumour tissue of each patient. Analysis of patient samples revealed other myeloid derived immune cells. The frequency of myeloid derived suppressor cells (CD45+CD33+CD11b+CD64-CD14-) was significantly increased (tumour = 0.64 ± 0.22, control bowel = 0.26 ± 0.07, P = 0.03, n = 7) in tumour tissue compared to control bowel. A large population of gut resident macrophages was observed in both tumour and control bowel tissue. These macrophages are highly phagocytic and unresponsive to cytokines – a phenotype that could be beneficial in restricting tumour growth. Here, this population could be subdivided into gut resident (CD45+CD33+CD14-CD11b-CD64-) and inflammatory gut resident (CD45+CD33+CD14+CD11b+CD64+) macrophages. Macrophages (CD45+CD64+CD11b+), sorted from both tumour and control bowel tissue, were cultured with T cells (CD3+) sorted from patient blood. Tumour macrophages increased T cell IFN-g and IL-17 production, as measured by flow cytometry, compared to T cell cultured alone, whereas control bowel macrophages did not.This study highlights the complexity of macrophages and provides methods to examine them ex vivo from human tissue. Data gained from this study could be used for future investigation of macrophages as both prognostic and therapeutic targets. Optogenetic activation of gonadotropin-releasing hormone neurons reveals minimal requirements for pulsatile luteinising hormone secretion. P Campos, A Herbison, B Hyland. Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.The reproductive system is critically dependent upon patterned pulsatile hormone release that is driven by the gonadotropin-releasing hormone (GnRH) neurons. The patterns of neuronal activity responsible for generating the pulsatile release of GnRH are unknown. We developed a methodology in mice for remotely controlling the activity of the GnRH neurons in vivo to establish the parameters of activation required to evoke a pulse of luteinising hormone (LH) secretion from the pituitary.Injections of Cre-dependent channelrhodopsin (ChR2)-bearing adeno-associated virus into the median eminence of GnRH-Cre mice resulted in the selective expression of ChR2 in GnRH neurons (93 ± 8 % double-labeled neurons, mean ± sem, n = 6, in the rostral preoptic area (rPOA)). Acute brain slice experiments demonstrated that ChR2-expressing GnRH neurons could be driven to fire with high spike fidelity with blue light stimulation frequencies up to 40 Hz. In vivo, optical fibers were implanted in the vicinity of GnRH neurons within the rPOA of anesthetised, ovariectomised mice. Optogenetic activation of GnRH neurons for 30 s to five min time periods over a range of different frequencies revealed that 10 Hz stimulation for two minutes was the minimum required to generate a pulse-like increment of LH (1.49 ± 0.08 fold increase, n = 8) which was significantly different (P < 0.001, one-way ANOVA) from control animals. Under these conditions, the dynamics of optogenetically-evoked LH secretion are very similar to that of endogenous LH pulses with the rise time and increment in LH being identical. This suggests that the minimal parameters of GnRH neuron activation reported here are likely to be that occurring in vivo for pulsatile LH secretion.This study provides the first major insight into the nature of GnRH neuron activation required to generate pulsatile LH secretion and provides the foundations for understanding pulsatility within the reproductive axis. Triazole drug target structures in a resistant yeast strain. A Sagatova1, M Keniya1, F Huschmann1,2, S Willbanks3, R Cannon1, J Tyndall2, B Monk1. 1Sir John Walsh Research Institute and Department of Oral Sciences, Faculty of Dentistry, 2School of Pharmacy, 3Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin.Fungal pathogens are estimated to be responsible for ~1.5 million deaths annually. Triazole drugs provide some of the most widely used treatments of fungal infections. They target the membrane protein lanosterol 14α-demethylase (Erg11p) which catalyses the rate limiting step of ergosterol biosynthesis. Inhibition of Erg11p depletes cell membranes of ergosterol and inhibits fungal growth. Resistance to triazoles can arise due to mutations in Erg11p that reduce binding affinity of the drug. An increasing incidence of resistance in fungal pathogens such as Candida species is of major concern. This project aims to understand how a mutation in Erg11p differentially affects the binding of triazoles.Wild type hexahistidine-tagged ERG11 or a mutant allele that translates to a single amino acid change found in clinical isolates of pathogenic fungi was cloned in a Saccharomyces cerevisiae membrane protein overexpression system. Endogenous ERG11 was deleted. Recombinant ScErg11p6×His was solubilised with N-decyl-β-D-maltoside and purified by Ni-NTA affinity chromatography and size exclusion chromatography. Purified enzyme was used for ligand binding assays and crystallographic studies. X-ray crystal structures of the ScErg11p Y140F mutant in complex with itraconazole (ITC) and fluconazole (FLC) were determined at a resolution of 2 Å from crystallographic data collected at the Australian Synchrotron.The Candida albicans Erg11p Y132F mutation gives resistance to FLC in clinical isolates. The equivalent Y140F mutation in ScErg11p conferred resistance to FLC but not ITC. The ScErg11p Y140F structure with the long-tailed triazole ITC revealed binding matching that of the wild-type, with an additional water molecule between a heme carboxyl and the aromatic side chain of F140. Comparison of wild type ScErg11p structure to the Y140F mutant in complex with FLC revealed a disruption of well-ordered hydrogen bonding interactions between the enzyme and the drug. Disruption of this hydrogen bonding network helps to explain FLC resistance of the mutant enzyme. Modulation of heart rate by specific β1- and β2-adrenoceptor stimulation in conscious Zucker type II Diabetic Fatty rats. R Cook, C Bussey, P Cragg, R Lamberts. Department of Physiology and HeartOtago, Otago School of Medical Sciences, University of Otago, Dunedin.Resting heart rate (HR) is the strongest predictor of mortality in patients with cardiac disease. The β-adrenoceptor (β-AR) functional responsiveness and β1-AR expression, key components of sinoatrial rate regulation, are reduced in many cardiomyopathies. An in vitro model suggests co-localisation of ‘funny'-channels and β2-ARs via lipid-rich caveolae is the mechanism through which HR is modulated. β2-AR redistribution in the failing heart compensates for the decreased β1-AR function. Whether similar β2-AR subtype changes contribute to HR dysfunction in diabetes is unknown. We aimed to determine the effects of specific β1- and β2-AR stimulation on HR in type II diabetes in vivo.Male Zucker Diabetic Fatty (ZDF) rats and lean littermates (20-week old, n = 8) were implanted with a radiotelemeter to measure arterial blood pressure and derive HR and a vascular access port to inject drugs intravenously. Cumulative doses of isoproterenol (non-selective β1- and β2-AR agonist, 0.001 – 3 µg.kg-1) or fenoterol (non-selective β2-AR agonist, 0.001 – 3 µg.kg-1) in the presence or absence of atenolol (selective β1-AR antagonist, 2000 µg.kg-1) were injected.Increases in HR due to non-selective β-AR stimulation were not significantly different between groups across doses (at 0.1 µg.kg-1: 106 ± 10 vs. 109 ± 13 bpm). However the HR response to specific β2-AR stimulation was significantly reduced in ZDF rats (25 ± 4 vs. 2 ± 4 bpm and 47 ± 7 vs. 20 ± 2 bpm; P < 0.002 at 0.03 and 0.1 µg.kg-1, respectively). HR responses adjusted to the baseline were modulated by β2-AR in controls although in ZDF rats HR regulation involved both β1- and β2-ARs.Thus, in conscious ZDF rats β-AR responsiveness was not affected, whereas the role of β1-ARs was increased. Therefore, HR regulation by β1- and β2-AR is altered in diabetes, and β1-AR signaling might play a compensatory role in maintaining β-AR reactivity. Polymerase-1 and transcript release factor and caveolae in prostate cancer; which is more important? J-Y Low, H Nicholson. Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.Prostate cancer poses a significant health threat to men world-wide. Caveolae are “cave-like” invaginations that are found in the plasma membrane. Polymerase-1 and transcript release factor (PTRF) is reported to be involved in the formation of caveolae. Expression of PTRF and caveolae are lost in prostate cancer cell lines and tissues while restoration of PTRF expression leads to formation of caveolae and reduced aggressive phenotypes in vitro and in vivo. However it is unclear whether the effect seen is due to PTRF itself or the restoration of caveolae. This study examines whether PTRF or caveolae are involved in prostate cancer progression.Using normal prostate epithelial cells, RWPE-1, PTRF was knocked down with 120 nM siRNA, with scrambled siRNA used as control, and caveolae were disrupted with 10 mM of methyl-β-cyclodextrin (MBCD). Transmission electron microscopy was performed to determine the number of caveolae. Cell proliferation was measured with CellTiter 96 aqueous non-radioactive cell proliferation kit. Migration and invasion assays were performed with Transwell migration and Matrigel invasion inserts respectively. Changes in cellular signalling were examined with Pathscan intracellular signalling array. Statistical analyses were computed with Student's unpaired t-test.Knock down PTRF, or MBCD treatment, reduced caveolae numbers in RWPE-1 cells (P < 0.05 siRNA; P < 0.01 MBCD, n = 50 cells). Following both treatments increased proliferation (P < 0.05, n = 6), migration (P < 0.05, n = 3) and invasion (P < 0.001 siRNA; P < 0.01 MBCD, n = 3) was observed. However, cell-signalling arrays showed a different pattern of signaling molecules activation. Two fold increase in p-STAT3 (P < 0.05, n = 4) was seen after knocking down PTRF, while disrupting caveolae resulted in two fold increase of p-ERK1/2 (P < 0.01, n = 4).Both PTRF and caveolae may be involved in prostate cancer progression, however these elements may drive prostate cancer progression through different signaling pathways. Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome. A Moore, M Prescott, R Campbell. Department of Physiology and Centre for Neuroendocrinology, Otago School of Medical Sciences, University of Otago, Dunedin.Polycystic ovarian syndrome (PCOS) is the most common cause of infertility among women of reproductive age. An increase in luteinising hormone (LH) pulse frequency associated with PCOS is suggestive of changes in gonadotropin-releasing hormone (GnRH) neuron regulation. We hypothesise that altered neuronal regulation of GnRH neurons is due to changes in their afferent synaptic input.PCOS was modeled in mice by prenatal androgen (PNA) exposure. GnRH-green fluorescent protein (GFP) mice were used to assess synaptic input to GnRH neurons in control and PNA-treated mice (n = 8 / group). Immunohistochemical labeling of brain sections revealed a significant increase in the density of vesicular GABA transporter (vGaT) appositions with GnRH neurons in PNA-treated mice (0.8 ± 0.08 appositions/μm) compared to controls (0.4 ± 0.02 appositions/μm, P < 0.05, two-tailed t-test). As GABA signaling to GnRH neurons can be excitatory, increased GABAergic input may elevate GnRH neuron activity. However, the identity of GABAergic neuronal populations regulating GnRH neuron activity remains largely unknown.To address whether increased GABA innervation of GnRH neurons originates in the arcuate nucleus (ARN), a Cre-dependent adenoviral vector expressing farnesylated enhanced GFP (EGFPf) was injected into the ARN of vGaT-Cre mice. Immunofluorescent labeling revealed that EGFPf-positive ARN GABAergic projections heavily contacted and even bundled with GnRH neuron dendrites. In control and PNA-treated mice, 61 ± 6.1 % and 60 ± 10.5 % of GnRH neurons closely apposed projections from ARN GABAergic neurons, respectively (n = 6 / group). Furthermore, the density of fibres apposing the GnRH neuron dendrite was even greater in PNA-treated mice (0.09 ± 0.02 apposing fibres/μm) compared to controls (0.04 ± 0.006 apposing fibres/μm, P < 0.05).These data describe a novel, robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome. \r\n

Summary

Abstract

Aim

Method

Results

Conclusion

Author Information

Acknowledgements

Correspondence

Correspondence Email

Competing Interests

Contact diana@nzma.org.nz
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