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    ABSTRACT: The neuroendocrine response to episodes of acute stress is crucial for survival whereas the prolonged response to chronic stress can be detrimental. Learning and memory are particularly susceptible to stress with cognitive deficits being well characterized consequences of chronic stress. Although there is good evidence that acute stress can enhance cognitive performance, the mechanism(s) for this are unclear. We find that hippocampal slices, either prepared from rats following 30 min restraint stress or directly exposed to glucocorticoids, exhibit an N-methyl-d-aspartic acid receptor-independent form of long-term potentiation. We demonstrate that the mechanism involves an NMDA receptor and PKA-dependent insertion of Ca(2+)-permeable AMPA receptors into synapses. These then trigger the additional NMDA receptor-independent form of LTP during high frequency stimulation.
    Brain 11/2013;
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    ABSTRACT: The paraventricular nucleus (PVN) of the hypothalamus plays an important role in the regulation of sympathetic nerve activity (SNA), which is significantly elevated in chronic heart failure (CHF). Fractalkine (FKN) and its cognate receptor, CX3CR1, are constitutively expressed in the central nervous system but its role and physiological significance are not well known. The aims of the present this study were to determine whether FKN plays a cardiovascular role within the PVN and to investigate how the actions of FKN this might be altered in CHF. Here we show that both FKN and CX3CR1 are expressed on neurons in the PVN of rats suggesting that it may play a physiological function in this brain nucleus. Unilateral microinjection of FKN, directly into the PVN of anaesthetised rats, elicited a significant dose-related decrease in blood pressure (1.0nmol, -5±3 mmHg; 2.5nmol, 13±2; 5.0nmol, 22±3 mmHg; 7.5nmol -32±3 mmHg) and a concomitant increase in heart rate (1.0nmol, 6±3 bpm; 2.5nmol, 11±3; 5nmol, 18±4 bpm; 7.5nmol 27±5 bpm) compared to control saline microinjections. In order to see if FKN signaling is altered in CHF rats we first performed qRT-PCR and western blot analysis and followed these experiments with functional studies in HF and sham-control rats. We found a significant increase in CX3CR1 mRNA and protein expression, as determined by quantitative RT-PCR and Western blot analysis respectively, in PVN of rats with CHF compared to sham control rats. We also found that the blood pressure effects of FKN (2.5 nmol in 50 nl) were significantly attenuated in rats with CHF (ΔMAP -6±3 mmHg) compared to sham-operated control (ΔMAP -16±6 mmHg) rats. These data suggest that FKN and its receptor, CX3CR1 modulates cardiovascular function at the level of the PVN and that its actions within this nucleus are altered in heart failure.
    Experimental physiology 09/2013;
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    ABSTRACT: Basal forebrain cholinergic neurons (bfCNs) which provide innervation to the hippocampus and cortex, are required for memory and learning, and are primarily affected in Alzheimer's Disease (AD), resulting in related cognitive decline. Therefore generation of a source of bfCNs from human pluripotent stem cells (hPSCs) is crucial for in vitro disease modeling and development of novel AD therapies. In addition, for the advancement of regenerative approaches there is a requirement for an accurate developmental model to study the neurogenesis and survival of this population. Here we demonstrate the efficient production of bfCNs, using a novel embryoid body (EB) based non-adherent differentiation (NAdD) protocol. We establish a specific basal forebrain neural stem cell (NSC) phenotype via expression of the basal forebrain transcription factors NKX2.1 and LHX8, as well as the general forebrain marker FOXG1. We present evidence that this lineage is achieved via recapitulation of embryonic events, with induction of intrinsic hedgehog signaling, through the use of a 3D non-adherent differentiation system. This is the first example of hPSC-derived basal forebrain-like NSCs, which are scalable via self-renewal in prolonged culture. Furthermore upon terminal differentiation these basal forebrain-like NSCs generate high numbers of cholinergic neurons expressing the specific markers ChAT, VACht and ISL1. These hPSC-derived bfCNs possess characteristics that are crucial in a model to study AD related cholinergic neuronal loss in the basal forebrain. Examples are expression of the therapeutic target p75(NTR), the release of acetylcholine, and demonstration of a mature, and functional electrophysiological profile. In conclusion, this work provides a renewable source of human functional bfCNs applicable for studying AD specifically in the cholinergic system, and also provides a model of the key embryonic events in human bfCN development.
    Stem Cell Research 08/2013; 11(3):1206-1221.
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    ABSTRACT: Botulinum neurotoxin is a potent inhibitor of acetylcholine secretion and acts by cleaving members of the soluble NSF-attachment protein receptor (SNARE) family which are critical to exocytotic vesicular secretion. However, the potential of botulinum neurotoxin for treating secretory disease is limited both by its neural selectivity and the necessity for direct injection into the relevant target tissue. To circumvent these limitations, a technology platform called targeted secretion inhibitors (TSI) is being developed. TSI are derived from botulinum neurotoxin but are retargeted to specific cell types to inhibit aberrant secretion. A TSI called qGHRH-LHN/D, with a growth hormone releasing hormone (GHRH) receptor targeting domain and designed to specifically inhibit pituitary somatotroph GH release through cleavage of the SNARE protein, vesicle associated membrane protein (VAMP), has recently been described. Here, we show this TSI activates GHRH receptors in primary cultured rat pituicytes, is internalised into these cells, depletes VAMP-3 and inhibits PMA-induced GH secretion. In vivo studies show that this TSI, but not one with an inactive catalytic unit, produces a dose-dependent inhibition of pulsatile growth hormone secretion, thus confirming its mechanism of action through VAMP cleavage. Selectivity of action has been shown by the lack of effect of this TSI in vivo on secretion from thyrotrophs, corticotrophs and gonadotrophs. In the absence of suitable in vivo models, these data provide proof of concept for the use of somatotroph-targeted TSI in the treatment of Acromegaly and moreover raise the potential that TSI could be used to target other diseases characterised by hypersecretion.
    Endocrinology 07/2013;
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    ABSTRACT: Tonic GABA(A) receptor-mediated signaling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K(+)-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased.
    The Journal of Physiology 02/2013;
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    ABSTRACT: The neuropeptide apelin is expressed in hypothalamic paraventricular and supraoptic nuclei and mediates its effects via activation of the apelin receptor (APJ). Evidence suggests a role for apelin and APJ in mediating the neuroendocrine response to stress. To understand the physiological role of APJ in regulation of the hypothalamic-pituitary-adrenal (HPA) axis, we measured adrenocorticotropic hormone (ACTH) and corticosterone (CORT) plasma levels in male and female mice lacking APJ (APJ KO) and in wild-type controls, in response to a variety of acute stressors. Exposure to mild restraint, systemic injection of lipopolysaccharide (LPS), insulin-induced hypoglycaemia, and forced swim (FS) stressors, elevated plasma ACTH and CORT levels in wild-type mice. Acute mild restraint significantly increased plasma ACTH and CORT to a similar level in APJ KO mice as in wild-type mice. However, an intact APJ was required for a conventional ACTH, but not CORT, response to LPS administration in male mice, and to insulin-induced hypoglycaemia in male and female mice. In contrast, APJ KO mice displayed an impaired CORT response to acute FS stress, regardless of gender. These data indicate that APJ has a role in regulation of the HPA axis response to some acute stressors and has a gender-specific function in peripheral immune activation of the HPA axis.
    Journal of Endocrinology 01/2013;
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    ABSTRACT: The circadian rhythm of corticosterone (CORT) secretion from the adrenal cortex is regulated by the suprachiasmatic nucleus (SCN), which is entrained to the light-dark cycle. Since the circadian CORT rhythm is associated with circadian expression of the steroidogenic acute regulatory (StAR) protein, we investigated the 24 h pattern of hormonal secretion (ACTH and CORT), steroidogenic gene expression (StAR, SF-1, DAX1 and Nurr77) and the expression of genes involved in ACTH signalling (MC2R and MRAP) in rats entrained to a normal light-dark cycle. We found that circadian changes in ACTH and CORT were associated with the circadian expression of all gene targets; with SF-1, Nurr77 and MRAP peaking in the evening, and DAX1 and MC2R peaking in the morning. Since disruption of normal SCN activity by exposure to constant light abolishes the circadian rhythm of CORT in the rat, we also investigated whether the AM-PM variation of our target genes was also disrupted in rats exposed to constant light conditions for 5 weeks. We found that the disruption of the AM-PM variation of ACTH and CORT secretion in rats exposed to constant light was accompanied by a loss of AM-PM variation in StAR, SF-1 and DAX1, and a reversed AM-PM variation in Nurr77, MC2R and MRAP. Our data suggest that circadian expression of StAR is regulated by the circadian expression of nuclear receptors and proteins involved in both ACTH signalling and StAR transcription. We propose that ACTH regulates the secretion of CORT via the circadian control of steroidogenic gene pathways that become dysregulated under the influence of constant light.
    Molecular and Cellular Endocrinology 11/2012;
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    ABSTRACT: Evidence suggests that the acquisition of recognition memory depends upon CREB-dependent long-lasting changes in synaptic plasticity in the perirhinal cortex.The CREB-responsive microRNA miR-132 has been shown to regulate synaptic transmission and we set out to investigate a role for this microRNA in recognition memory and its underlying plasticity mechanisms. To this end we mediated the specific overexpression of miR-132 selectively in the rat perirhinal cortex and demonstrated impairment in short-term recognition memory. This functional deficit was associated with a reduction in both long-term depression and long-term potentiation. These results confirm that microRNAs are key coordinators of the intracellular pathways that mediate experience-dependent changes in the brain. In addition, these results demonstrate a role for miR-132 in the neuronal mechanisms underlying the formation of short-term recognition memory.
    European Journal of Neuroscience 07/2012; 36(7):2941-8.
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    ABSTRACT: The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the AP-1 gene family, namely c-fos, c-jun, fosl1 and junD. As JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and quantitative RT-PCR to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24-hours and 72-hours of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double staining fluorescence immunohistochemistry with a neurone specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin containing neurones with markedly less staining seen in oxytocin containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members. © 2012 The Authors. Journal of Neuroendocrinology © 2012 Blackwell Publishing Ltd.
    Journal of Neuroendocrinology 07/2012;
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    ABSTRACT: Plasma levels of corticosterone exhibit both circadian and ultradian rhythms. The circadian component of these rhythms is regulated by the suprachiasmatic nucleus (SCN). Our studies investigate the importance of the SCN in regulating ultradian rhythmicity. Two approaches were used to dissociate the hypothalamic-pituitary-adrenal (HPA) axis from normal circadian input in rats: (i) exposure to a constant light (LL) environment and (ii) electrolytic lesioning of the SCN. Blood was sampled using an automated sampling system. As expected, both treatments resulted in a loss of the circadian pattern of corticosterone secretion. Ultradian pulsatile secretion of corticosterone however, was maintained across the 24 h in all animals. Furthermore, the loss of SCN input revealed an underlying relationship between locomotor and HPA activity. In control (LD) rats there was no clear correlation between ultradian locomotor activity and hormone secretion, whereas, in LL rats, episodes of ultradian activity were consistently followed by periods of increased pulsatile hormone secretion. These data clearly demonstrate that the ultradian rhythm of corticosterone secretion is generated through a mechanism independent of the SCN input, supporting recent evidence for a sub-hypothalamic pulse generator.
    European Journal of Neuroscience 07/2012; 36(8):3142-50.
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