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    ABSTRACT: Voltage gated sodium channel blockers are not traditionally recommended for osteoarthritis (OA) pain therapy, but given the large peripheral drive that follows OA development there is a rationale for their use. Using a rat model of monosodium iodoacetate (MIA) induced osteoarthritis we used in vivo electrophysiology to assess the effects of the Nav1.7 and Nav 1.8 selective antagonists, ProTxII and A-803467 respectively, on the evoked activity of spinal dorsal horn neurons in response to electrical, mechanical and thermal stimuli applied to the peripheral receptive field. These studies allow examination of the roles of these channels in suprathreshold stimuli, not amenable to behavioural threshold measures. Spinal administration of ProTxII significantly reduced neuronal responses evoked by mechanical punctate (von Frey (vF) 8- 60g) and noxious thermal (45 and 48 (0)C) stimuli in MIA rats only. A-803467 significantly inhibited neuronal responses evoked by vF 8 - 60g and 48(0)C heat after spinal administration; significantly inhibited responses evoked by brush, vFs 26-60g and 40- 48(0)C stimuli after systemic administration; significantly inhibited the electrically evoked Aδ-, C-fiber, post-discharge, Input and wind-up responses and the brush, vFs 8- 60g and 45 - 48(0)C evoked neuronal responses after intra plantar injection in the MIA group. In comparison A-803467 effects in the sham group were minimal and included a reduction of the neuronal response evoked by vF60g and 45(0)C heat stimulation after spinal administration, no effect after systemic administration and an inhibition of the evoked response to 45(0)C heat after intra plantar injection only. The observed selective inhibitory effect of ProTxII and A-803467 for the MIA treated group suggest an increased role of Nav 1.7 and 1.8 within nociceptive pathways in the arthritic condition, located at peripheral and central sites. These findings demonstrate the importance of, and add to, the mechanistic understanding of these channels in osteoarthritic pain. Copyright © 2015. Published by Elsevier Ltd.
    Neuroscience 03/2015; 15. DOI:10.1016/j.neuroscience.2015.03.042
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    ABSTRACT: The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labeling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β-α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus.
    Neuropharmacology 01/2015; 20. DOI:10.1016/j.neuropharm.2015.01.001
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    ABSTRACT: Treatments for neuropathic pain are either not fully effective or have problematic side effects. Combinations of drugs are often used. Tapentadol is a newer molecule that produces analgesia in various pain models through two inhibitory mechanisms, namely central μ-opioid receptor (MOR) agonism and noradrenaline reuptake inhibition. These two components interact synergistically, resulting in levels of analgesia similar to opioid analgesics such as oxycodone and morphine, but with more tolerable side effects. The right central nucleus of the amygdala (CeA) is critical for the lateral spinal ascending pain pathway, regulates descending pain pathways and is key in the emotional-affective components of pain. Few studies have investigated the pharmacology of limbic brain areas in pain models. Here we determined the actions of systemic tapentadol on right CeA neurones of animals with neuropathy and which component of tapentadol contributes to its effect. Neuronal responses to multimodal peripheral stimulation of animals with spinal nerve ligation or sham surgery were recorded before and after two doses of tapentadol. After the higher dose of tapentadol either naloxone or yohimbine were administered. Systemic tapentadol resulted in dose-dependent decrease in right CeA neuronal activity only in neuropathy. Both naloxone and yohimbine reversed this effect to an extent that was modality selective. The interactions of the components of tapentadol are not limited to the synergy between the MOR and α2-adrenoceptors seen at spinal levels, but are seen at this supraspinal site where suppression of responses may relate to the ability of the drug to alter affective components of pain.
    European Journal of Pharmacology 01/2015; 7. DOI:10.1016/j.ejphar.2014.11.032
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    ABSTRACT: The slow afterhyperpolarizing current (sIAHP ) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP , resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP , suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing camp and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP . Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP , underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons. © 2013 Wiley Periodicals, Inc.
    Hippocampus 01/2014; 24(1). DOI:10.1002/hipo.22201
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    ABSTRACT: Tonic inhibitory conductances mediated by GABAA receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABAA conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current.
    Frontiers in Neural Circuits 12/2013; 7:193. DOI:10.3389/fncir.2013.00193
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    ABSTRACT: The strength of the placebo effect is influenced by social contexts and individual personality. Although facial expressions provide important contextual cues no studies on their influence on the placebo response has been performed hitherto. Here we tested whether (1) the observation of facial expressions with different emotional content (Neutral, Pain and Happy) affects the magnitude of placebo analgesia, and (2) inter-individual differences in personality traits interact with any modulation of placebo response induced by facial expression. Twenty-seven healthy participants underwent classical placebo conditioning, and subsequently rated the intensity and unpleasantness of their subjective experience of pain elicited by nociceptive-specific laser pulses delivered to the right hand dorsum. On each trial, different visual cues signaled the occurrence of a laser stimulus alone or of a laser stimulus accompanied by a sham analgesic procedure. In the conditioning period, cues signaling the sham analgesic treatment were followed by laser stimuli whose intensity was surreptitiously lowered. In the test period, either cue was followed by laser stimuli of the same intensity. The observation of facial expressions with different emotional content enhanced significantly the placebo analgesia. In particular, a significantly greater analgesic effect was observed when facial expressions with emotional content were presented concomitantly to the nociceptive stimulation. The enhancement of placebo analgesia during the observation of facial expressions was not correlated with personality traits like empathy and behavioural activation/inhibition. These findings quantify for the first time the effect of facial expressions on the magnitude of placebo analgesia.
    Pain 12/2013; 155(4). DOI:10.1016/j.pain.2013.11.021
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    ABSTRACT: The α2δ-1 protein is an auxiliary subunit of voltage-gated calcium channels, critical for neurotransmitter release. It is up-regulated in dorsal root ganglion (DRG) neurons following sensory nerve injury, and is also the therapeutic target of the gabapentinoid drugs, which are efficacious in both experimental and human neuropathic pain conditions. α2δ-1 has three spliced regions, A, B and C. A and C are cassette exons, whereas B is introduced via an alternative 3' splice acceptor site. Here we have examined the presence of α2δ-1 splice variants in DRG neurons, and have found that although the main α2δ-1 splice variant in DRGs is the same as that in brain (α2δ-1 ΔA+B+C), there is also another α2δ-1 splice variant (ΔA+BΔC), which is expressed in DRG neurons and is differentially up-regulated compared to the main DRG splice variant α2δ-1 ΔA+B+C following spinal nerve ligation. Furthermore, this differential up-regulation occurs preferentially in a small non-myelinated DRG neuron fraction, obtained by density gradient separation. The α2δ-1 ΔA+BΔC splice variant supports CaV2 calcium currents with unaltered properties compared to α2δ-1 ΔA+B+C, but shows a significantly reduced affinity for gabapentin. This variant could therefore play a role in determining the efficacy of gabapentin in neuropathic pain.
    Pain 12/2013; 155(3). DOI:10.1016/j.pain.2013.12.001
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    ABSTRACT: We aimed to develop and validate a reliable method for stable long-term recordings of EEG activity in zebrafish, which is less prone to artifacts than current invasive techniques. EEG activity was recorded with a blunt electrolyte-filled glass pipette placed on the zebrafish head mimicking surface EEG technology in man. In addition, paralysis of agarose-embedded fish using D-tubocurarine excluded movement artifacts associated with epileptic activity. This non-invasive recording technique allowed recordings for up to one hour and produced less artifacts than impaling the zebrafish optic tectum with a patch pipette. Paralyzed fish survived, and normal heartbeat could be monitored for over 1h. Our technique allowed the demonstration of specific epileptic activity in kcnj10a morphant fish (a model for EAST syndrome) closely resembling epileptic activity induced by pentylenetetrazol. This new method documented that seizures in the zebrafish EAST model were ameliorated by pentobarbitone, but not diazepam, validating its usefulness. In conclusion, non-invasive recordings in paralyzed EAST syndrome zebrafish proved stable, reliable and robust, showing qualitatively similar frequency spectra to those obtained from pentylenetetrazol-treated fish. This technique may prove particularly useful in zebrafish epilepsy models that show infrequent or conditional seizure activity.
    PLoS ONE 11/2013; 8(11):e79765. DOI:10.1371/journal.pone.0079765
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    ABSTRACT: Epigenetic modifications of chromatin represent a fundamental mechanism by which eukaryotic cells adapt their transcriptional response to developmental and environmental cues. Although an increasing number of molecules have been linked to epigenetic changes, the intracellular pathways that lead to their activation/repression have just begun to be characterized. Here, we demonstrate that inositol hexakisphosphate kinase 1 (IP6K1), the enzyme responsible for the synthesis of the high-energy inositol pyrophosphates (IP7), is associated with chromatin and interacts with Jumonji domain containing 2C (JMJD2C), a recently identified histone lysine demethylase. Reducing IP6K1 levels by RNAi or using mouse embryonic fibroblasts derived from ip6k1(-/-) knockout mice results in a decreased IP7 concentration that epigenetically translates to reduced levels of trimethyl-histone H3 lysine 9 (H3K9me3) and increased levels of acetyl-H3K9. Conversely, expression of IP6K1 induces JMJD2C dissociation from chromatin and increases H3K9me3 levels, which depend on IP6K1 catalytic activity. Importantly, these effects lead to changes in JMJD2C-target gene transcription. Our findings demonstrate that inositol pyrophosphate signaling influences nuclear functions by regulating histone modifications.
    Proceedings of the National Academy of Sciences 11/2013; 110(47). DOI:10.1073/pnas.1309699110
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    ABSTRACT: Two-photon microscopy is widely used to study brain function, but conventional microscopes are too slow to capture the timing of neuronal signalling and imaging is restricted to one plane. Recent development of acousto-optic-deflector-based random access functional imaging has improved the temporal resolution, but the utility of these technologies for mapping 3D synaptic activity patterns and their performance at the excitation wavelengths required to image genetically-encoded indicators have not been investigated. New Method. Here, we have used a compact acousto-optic lens (AOL) two-photon microscope to make high speed [Ca(2+)] measurements from spines and dendrites distributed in 3D with different excitation wavelengths (800nm - 920nm). We show simultaneous monitoring of activity from many synaptic inputs distributed over the 3D arborisation of a neuronal dendrite using both synthetic as well as genetically-encoded indicators. We confirm the utility of AOL-based imaging for fast in vivo recordings by measuring, simultaneously, visually-evoked responses in 100 neurons distributed over a 150μm focal depth range. Moreover, we explore ways to improve the measurement of timing of neuronal activation by choosing specific regions within the cell soma. Comparison with existing Methods. These results establish that AOL-based 3D random access two-photon microscopy has a wider range of neuroscience applications than previously shown. Our findings show that the compact AOL microscope design has the speed, spatial resolution, sensitivity and wavelength flexibility to measure 3D patterns of synaptic and neuronal activity on individual trials.
    Journal of neuroscience methods 11/2013; 222(100). DOI:10.1016/j.jneumeth.2013.10.021
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