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O'Connor, J. J.
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O'Connor, J. J.
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O'Connor, J. J.
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Now showing 1 - 10 of 22
- PublicationAutomated analysis of intracellular calcium fluorescence in rat organotypic hippocampal cultures: comparison to a manual, observer based method.(Elsevier, 2014-02)
; ; ; ; The technical advances made in microscopy have been matched by an increase in the application of fluorescent microscopy to answer scientific questions. While analysis of fluorescent microscopy images represents a powerful tool, one must be aware of the potential pitfalls. Frequently, the analysis methods applied involve at least some manual steps which are dependent on an observers input. Typically these steps are laborious and time consuming, but more importantly they are also influenced by an individual observer¿s bias, drift or imprecision. This raises concerns about the repeatability and definitiveness of the reported observations. Using calcium fluorescence in organotypic hippocampal slices as an experimental platform, we demonstrate the influence that manual interventions can exert on an analysis. We show that there is a high degree of variability between observers, and that this can be sufficient to affect the outcome of an experiment. To counter this, and to eliminate the disagreement between observers, we describe an alternative fully automated method which was created using EBImage package for R. This method has the added advantage of being fully open source and customisable, allowing for this approach to be applied to other analyses.379Scopus© Citations 2 - PublicationHypoxia-inducible factor signaling mechanisms in the central nervous systemIn the CNS neurons are highly sensitive to the availability of oxygen. In conditions where oxygen availability is decreased neuronal function can be altered, leading to injury and cell death. Hypoxia has been implicated in a number of central nervous system pathologies including stroke, head trauma, and neurodegenerative diseases. Depending on the duration and severity of the oxygen deprivation, cellular oxygen-sensor responses activate a variety of short- and long-term energy saving and cellular protection mechanisms. Failure of synaptic transmission can be observed within minutes following this hypoxia. The acute affects of hypoxia on synaptic transmission are primarily mediated by altering ion fluxes across membranes, presynaptic effects of adenosine and other actions at glutamatergic receptors. A more long-term feature of the response of neurons to hypoxia is the activation of transcription factors such as hypoxia inducible factor. The activation of hypoxia inducible factor is governed by a family of dioxygenases called hypoxia inducible factor prolyl 4 hydroxylases (PHDs). Under hypoxic conditions, PHD activity is inhibited, thereby allowing hypoxia inducible factor to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Inhibition of PHD activity stabilizes hypoxia inducible factor and other proteins thus acting as a neuroprotective agent. This review will focus on the response of neuronal cells to hypoxia inducible factor and its targets, including the prolyl hydroxylases. We also present evidence for acute effects of PHD inhibition on synaptic transmission and plasticity in the hippocampus.
881Scopus© Citations 35 - PublicationFrequency-dependent modulation of dopamine release by nicotine and dopamine D1 ligands: an in vitro fast cyclic voltammetry study in rat striatumNicotine is a highly addictive drug and exerts this effect partially through the modulation of dopamine release and increasing extracellular dopamine in regions such as the brain reward systems. Nicotine acts in these regions on nicotinic acetylcholine receptors. The effect of nicotine on the frequency dependent modulation of dopamine release is well established and the purpose of this study was to investigate whether dopamine D1 receptor (D1R) ligands have an influence on this. Using fast cyclic voltammetry and rat corticostriatal slices, we show that D1R ligands are able to modulate the effect of nicotine on dopamine release. Nicotine (500nM) induced a decrease in dopamine efflux at low frequency (single pulse or 5 pulses at 10Hz) and an increase at high frequency (100Hz) electrical field stimulation. The D1R agonist SKF-38393, whilst having no effect on dopamine release on its own or on the effect of nicotine upon multiple pulse evoked dopamine release, did significantly prevent and reverse the effect of nicotine on single pulse dopamine release. Interestingly similar results were obtained with the D1R antagonist SCH-23390. In this study we have demonstrated that the modulation of dopamine release by nicotine can be altered by D1R ligands, but only when evoked by single pulse stimulation, and are likely working via cholinergic interneuron driven dopamine release.
152Scopus© Citations 7 - PublicationOpioid mediated activity and expression of mu and delta opioid receptors in isolated human term non-laboring myometrium(Elsevier, 2013-01-05)
; ; ; ; The existence of opioid receptors in mammalian myometrial tissue is now widely accepted. Previously enkephalin degrading enzymes have been shown to be elevated in pregnant rat uterus and a met-enkephalin analogue has been shown to alter spontaneous contractility of rat myometrium. Here we have undertaken studies to determine the effects of met-enkephalin on in vitro human myometrial contractility and investigate the expression of opioid receptors in pregnant myometrium. Myometrial biopsies were taken from women undergoing elective caesarean delivery at term. Organ bath experiments were used to investigate the effect of the met-enkephalin analogue [d-Ala 2, d-met 5] enkephalin (DAMEA) on spontaneous contractility. A confocal immunofluorescent technique and real time PCR were used to determine the expression of protein and mRNA, respectively for two opioid receptor subtypes, mu and delta. DAMEA had a concentration dependent inhibitory effect on contractile activity (1×10−7 M–1×10−4 M; 54% reduction in contractile activity, P<0.001 at 1×10−4 M concentration). Mu and delta opioid receptor protein sub-types and their respective mRNA were identified in all tissues sampled. This is the first report of opioid receptor expression and of an opioid mediated uterorelaxant action in term human non-labouring myometrium in vitro942Scopus© Citations 7 - PublicationA role for tumor necrosis factor-alpha in ischemia and ischemic preconditioningDuring cerebral ischemia, elevation of TNF-α and glutamate to pathophysiological levels may induce dysregulation of normal synaptic processes, leading ultimately to cell death. Previous studies have shown that patients subjected to a mild transient ischemic attack within a critical time window prior to a more severe ischemic episode may show attenuation in the clinical severity of the stroke and result in a more positive functional outcome. Studies with organotypic hippocampal cultures and mixed primary hippocampal cultures have shown that prior incubation with low concentrations of glutamate and TNF-α increase the resistance of neurones to a subsequent insult from glutamate, AMPA and NMDA, while co-exposure of TNF-α and for example AMPA may have neuroprotective effects compared to cultures exposed to excitotoxic agents alone. In addition our work has shown that although glutamate and TNF-α pretreatment induces analogous levels of desensitisation of the intracellular calcium dynamics of neurons under resting conditions and in response to acute glutamate stimulation, their downstream signalling pathways involved in this response do not converge. Glutamate and TNF-α would appear to have opposing effects on resting Ca2+ levels which supports the proposal that they have distinct modes of preconditioning
136Scopus© Citations 72 - PublicationA role for adrenergic receptors in the uterotonic effects of ergometrine in isolated human term non-laboring myometrium(Lippincott, Williams and Wilkins, 2017-02)
; ; ; ; ; ; ; Background: Ergometrine is a uterotonic agent that is recommended in the prevention and management of post partum hemorrhage. Despite its long-standing use the mechanism by which it acts in humans has never been fully elucidated. The objective of this study was to investigate the role of adrenoreceptors in ergometrine's mechanism of action in human myometrium. The study examined the hypothesis that alpha adrenoreceptor antagonism would result in the reversal of the uterotonic effects of ergometrine. Methods: Myometrial samples were obtained from women undergoing elective cesarean delivery. The samples were then dissected into strips and mounted in organ bath chambers. Following generation of an ergometrine concentration-response curve (10-15 to 10-5 M), strips were treated with increasing concentrations of ergometrine (10-15 to 10-7 M) alone and ergometrine (10-7 to 10-5 M) in the presence of phentolamine (10-7 M), prazosin (10-7 M), propranolol (10-6 M) or yohimbine (10-6 M). The effects of adding ergometrine and the effect of drug combinations were analysed using linear mixed effects models with measures of amplitude (g), frequency (contractions/10min) and motility index (g*contractions/10min). Results: A total of 157 experiments were completed on samples obtained from 33 women. There was a significant increase in the motility index (adding 0.342 g*counts/10min/µM; 95% CI from 0.253 to 0.431, P<0.001), amplitude (0.078 g/µM; 95% CI, from 0.0344 to 0.121, P=5e-04) and frequency (0.051 counts/10min/µM; 95% CI, 0.038 to 0.063, P<0.001) in the presence of ergometrine. The α adrenergic antagonist phentolamine and the more selective α1 adrenergic antagonist prazosin, inhibited the ergometrine mediated increase in motility index, amplitude and frequency (-1.63 g*counts/10mins/µM and -16.70 g*counts/10mins/µM for motility index, respectively). Conclusions: These results provide novel evidence for a role for α adrenergic signaling mechanisms in the action of ergometrine on human myometrial smooth muscle in the in vitro setting. Information that sheds light on the mechanism of action of ergometrine may have implications for the development of further uterotonic agents.545Scopus© Citations 9 - PublicationTheta-Burst Stimulation of Hippocampal Slices Induces Network-Level Calcium Oscillations and Activates Analogous Gene Transcription to Spatial Learning(Public Library of Science, 2014-06-20)
; ; ; ; Over four decades ago, it was discovered that high-frequency stimulation of the dentate gyrus induces long-term potentiation (LTP) of synaptic transmission. LTP is believed to underlie how we process and code external stimuli before converting it to salient information that we store as 'memories'. It has been shown that rats performing spatial learning tasks display theta-frequency (3–12 Hz) hippocampal neural activity. Moreover, administering theta-burst stimulation (TBS) to hippocampal slices can induce LTP. TBS triggers a sustained rise in intracellular calcium [Ca2+]i in neurons leading to new protein synthesis important for LTP maintenance. In this study, we measured TBS-induced [Ca2+]i oscillations in thousands of cells at increasing distances from the source of stimulation. Following TBS, a calcium wave propagates radially with an average speed of 5.2 µm/s and triggers multiple and regular [Ca2+]i oscillations in the hippocampus. Interestingly, the number and frequency of [Ca2+]i fluctuations post-TBS increased with respect to distance from the electrode. During the post-tetanic phase, 18% of cells exhibited 3 peaks in [Ca2+]i with a frequency of 17 mHz, whereas 2.3% of cells distributed further from the electrode displayed 8 [Ca2+]i oscillations at 33 mHz. We suggest that these observed [Ca2+]i oscillations could lead to activation of transcription factors involved in synaptic plasticity. In particular, the transcription factor, NF-κB, has been implicated in memory formation and is up-regulated after LTP induction. We measured increased activation of NF-κB 30 min post-TBS in CA1 pyramidal cells and also observed similar temporal up-regulation of NF-κB levels in CA1 neurons following water maze training in rats. Therefore, TBS of hippocampal slice cultures in vitro can mimic the cell type-specific up-regulations in activated NF-κB following spatial learning in vivo. This indicates that TBS may induce similar transcriptional changes to spatial learning and that TBS-triggered [Ca2+]i oscillations could activate memory-associated gene expression.284Scopus© Citations 9 - PublicationAcute hypoxic exposure and prolyl-hydroxylase inhibition improves synaptic transmission recovery time from a subsequent hypoxic insult in rat hippocampus(Elsevier, 2018-12-15)
; ; ; ; In the CNS short episodes of acute hypoxia can result in a decrease in synaptic transmission which may be fully reversible upon re-oxygenation. Stabilization of hypoxia-inducible factor (HIF) by inhibition of prolyl hydroxylase domain (PHD) enzymes has been shown to regulate the cellular response to hypoxia and confer neuroprotection both in vivo and in vitro. Hypoxic preconditioning has become a novel therapeutic target to induce neuroprotection during hypoxic insults. However, there is little understanding of the effects of repeated hypoxic insults or pharmacological PHD inhibition on synaptic signalling. In this study we have assessed the effects of hypoxic exposure and PHD inhibition on synaptic transmission in the rat CA1 hippocampus. Field excitatory postsynaptic potentials (fEPSPs) were elicited by stimulation of the Schaffer collatoral pathway. 30 min hypoxia (gas mixture 95% N2/5% CO2) resulted in a significant and fully reversible decrease in fEPSP slope associated with decreases in partial pressures of tissue oxygen. 15-30 min of hypoxia was sufficient to induce stabilization of HIF in hippocampal slices. Exposure to a second hypoxic insult after 60 min resulted in a similar depression of fEPSP slope but with a significantly greater rate of recovery of the fEPSP. Prior single treatment of slices with the PHD inhibitor, dimethyloxalylglycine (DMOG) also resulted in a significantly greater rate of recovery of fEPSP post hypoxia. These results suggest that hypoxia and ‘pseudohypoxia’ preconditioning may improve the rate of recovery of hippocampal neurons to a subsequent acute hypoxia.405Scopus© Citations 5 - PublicationEffects of prolyl-hydroxylase inhibition and chronic intermittent hypoxia on synaptic transmission and plasticity in the rat CA1 and dentate gyrusChronic intermittent hypoxia (CIH) is an underlying component of obstructive sleep apnoea and has been shown to have deleterious and damaging effects on central neurons and to impair synaptic plasticity in the CA1 region of the rat hippocampus. CIH is a potent inducer of hypoxia inducible factor (HIF), a key regulator in a cell's adaptation to hypoxia that plays an important role in the fate of neurons during ischemia. Levels of HIF-1α are regulated by the activity of a group of enzymes called HIF-prolyl 4-hydroxylases (PHDs) and these have become potential pharmalogical targets for preconditioning against ischemia. However, little is known about the effects of prolyl hydroxylase inhibition and CIH on synaptic transmission and plasticity in sub-regions of the hippocampus. Male Wistar rats were treated for 7-days with either saline, CIH or PHD inhibition (dimethyloxaloylglycine, DMOG; 50mg/kg, i.p.). At the end of treatment all three groups showed no change in synaptic excitability or paired pulse paradigms. However long-term potentiation (LTP) was impaired in the CA1 region of the hippocampus of both CIH and DMOG treated animals. LTP induced in the dentate gyrus was not significantly affected by CIH or DMOG treatment. We also investigated the effect of 7-day CIH and DMOG treatment on the recovery of synaptic transmission following an acute 30 min hypoxic insult. CIH treated animals showed an improved rate of recovery of synaptic transmission following re-oxygentation in both the CA1 and dentate gyrus. This effect was not seen with 7-day DMOG treatment. These results suggest that LTP induction in the CA1 region is more sensitive to both CIH and DMOG treatment than the dentate gyrus.
855Scopus© Citations 32 - PublicationA brain-derived neurotrophic factor mimetic is sufficient to restore cone photoreceptor visual function in an inherited blindness model(Springer, 2017-09-12)
; ; ; ; ; ; ; ; ; ; Controversially, histone deacetylase inhibitors (HDACi) are in clinical trial for the treatment of inherited retinal degeneration. Utilizing the zebrafish dyeucd6 model, we determined if treatment with HDACi can rescue cone photoreceptor-mediated visual function. dye exhibit defective visual behaviour and retinal morphology including ciliary marginal zone (CMZ) cell death and decreased photoreceptor outer segment (OS) length, as well as gross morphological defects including hypopigmentation and pericardial oedema. HDACi treatment of dye results in significantly improved optokinetic (OKR) (~43 fold, p < 0.001) and visualmotor (VMR) (~3 fold, p < 0.05) responses. HDACi treatment rescued gross morphological defects and reduced CMZ cell death by 80%. Proteomic analysis of dye eye extracts suggested BDNF-TrkB and Akt signaling as mediators of HDACi rescue inour dataset. Cotreatment with the TrkB antagonist ANA-12 blocked HDACi rescue of visual function and associated Akt phosphorylation. Notably, sole treatment with a BDNF mimetic, 7,8-dihydroxyflavone hydrate, significantly rescued dye visual function (~58 fold increase in OKR, p < 0.001, ~3 fold increase in VMR, p < 0.05). In summary, HDACi and a BDNF mimetic are sufficient to rescue retinal cell death and visual function in a vertebrate model of inherited blindness.550Scopus© Citations 22