Research Output
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Publication
Theta-Burst Stimulation of Hippocampal Slices Induces Network-Level Calcium Oscillations and Activates Analogous Gene Transcription to Spatial Learning
2014-06-20, Sheridan, Graham K., Moeendarbary, Emad, Pickering, Mark, O'Connor, J. J., Murphy, Keith J.
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.
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Publication
Automated analysis of intracellular calcium fluorescence in rat organotypic hippocampal cultures: comparison to a manual, observer based method.
2014-02, Watters, Orla, Pickering, Mark, Murphy, Robert, Murphy, Keith J., O'Connor, J. J.
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.
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Publication
Preconditioning effects of tumor necrosis factor-α and glutamate on calcium dynamics in rat organotypic hippocampal cultures
2011-05, Watters, Orla, Pickering, Mark, O'Connor, J. J.
During cerebral ischemia, elevation of TNF-α and glutamate to pathophysiological levels in the hippocampus 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. In this study we have investigated the individual contribution of pre-exposure to TNF-α or glutamate in the development of ‘ischemic tolerance’ to a subsequent insult, using organotypic hippocampal cultures. At 6 days in vitro (DIV), cultures were exposed to an acute concentration of glutamate (30 μM) or TNF-α (5 ng/ml) for 30 min, followed by 24 h recovery period. We then examined the effect of the pretreatments on calcium dynamics of the cells within the CA region. We found that pretreatment with TNF-α or glutamate caused in a significant reduction in subsequent glutamate-induced Ca2+ influx 24 h later (control: 100.0 ± 0.8%, n = 7769 cells; TNF-α: 76.8 ± 1.0%, n = 5543 cells; glutamate: 75.3 ± 1.4%, n = 3859 cells; p < 0.001). Antagonism of circulating TNF-α (using infliximab, 25 μg/ml), and inhibition of the p38 MAP kinase pathway (using SB 203580, 10 μM) completely reversed this effect. However glutamate preconditioning did not appear to be mediated by p38 MAP kinase signalling, or NMDAR activation as neither SB 203580 nor D-AP5 (100 μM) altered this effect. Glutamate and TNF-α preconditioning resulted in small yet significant alterations in resting Ca2+ levels (control: 100.0 ± 0.9%, n = 2994 cells; TNF-α: 109.7 ± 1.0%, n = 2884 cells; glutamate; 93.3 ± 0.8%, n = 2899 cells; p < 0.001), TNF-α's effect reversed by infliximab and SB 203580. Both TNF-α and glutamate also resulted in the reduction of the proportion (P) of responsive cells within the CA region of the hippocampus (control; P = 0.459, 0.451 ≤ x ≥ 0.467, n = 14,968 cells, TNF-α; P = 0.40, 0.392 ≤ x ≥ 0.407, n = 15,218; glutamate; P = 0.388, 0.303 ≤ x≥ 0.396, n = 13,919 cells), and in the depression of the frequency of spontaneous Ca2+ events (vs. control: TNF-α: p > 0.00001, D = 0.0454; glutamate: p > 0.0001, D = 0.0534). Our results suggest that attenuation in resting Ca2+ activity and Ca2+ related responsiveness of cells within the CA region as a result of glutamate or TNF-α pre-exposure, may contribute to the development of ischemic tolerance.
Publication
CX3CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity
2014-08-12, Sheridan, Graham K., Wdowicz, Anita, Pickering, Mark, Watters, Orla, Halley, Paul G., O'Sullivan, Niamh C., Mooney, Claire, O'Connell, David J., O'Connor, J. J., Murphy, Keith J.
Several cytokines and chemokines are now known to play normal physiological roles in the brain where they act as key regulators of communication between neurons, glia, and microglia. In particular, cytokines and chemokines can affect cardinal cellular and molecular processes of hippocampal-dependent long-term memory consolidation including synaptic plasticity, synaptic scaling and neurogenesis. The chemokine, CX3CL1 (fractalkine), has been shown to modulate synaptic transmission and long-term potentiation (LTP) in the CA1 pyramidal cell layer of the hippocampus. Here, we confirm widespread expression of CX3CL1 on mature neurons in the adult rat hippocampus. We report an up-regulation in CX3CL1 protein expression in the CA1, CA3 and dentate gyrus (DG) of the rat hippocampus 2 h after spatial learning in the water maze task. Moreover, the same temporal increase in CX3CL1 was evident following LTP-inducing theta-burst stimulation in the DG. At physiologically relevant concentrations, CX3CL1 inhibited LTP maintenance in the DG. This attenuation in dentate LTP was lost in the presence of GABAA receptor/chloride channel antagonism. CX3CL1 also had opposing actions on glutamate-mediated rise in intracellular calcium in hippocampal organotypic slice cultures in the presence and absence of GABAA receptor/chloride channel blockade. Using primary dissociated hippocampal cultures, we established that CX3CL1 reduces glutamate-mediated intracellular calcium rises in both neurons and glia in a dose dependent manner. In conclusion, CX3CL1 is up-regulated in the hippocampus during a brief temporal window following spatial learning the purpose of which may be to regulate glutamate-mediated neurotransmission tone. Our data supports a possible role for this chemokine in the protective plasticity process of synaptic scaling.