Abstracts - 'Ion Channels in Health and Disease: a

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29/05/2018

Cambridge Neuroscience Abstracts :: Cambridge Neuroscience

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Abstracts - ‘Ion Channels in Health and Disease: a symposium to celebrate the 50th anniversary of the Nobel Prize awarded to Alan Hodgkin and Andrew Huxley’ : School of Life and Medical Sciences, University of Hertfordshire, UK Presenting author: Mr Yousif Shamsaldeen ([email protected]) Authors: Yousif Shamsaldeen, Lisa Lione, Chris Benham

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Authors' affiliations: School of Life and Medical Sciences, University of Hertfordshire Keywords: Neuropharmacology, Diabetes, Vascular dysfunction, Methylglyoxal, NO, NOS

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According to UK statistical data issued in April 2012, approximately 8% of the world population are diabetic from which 90% are diagnosed with type 2 diabetes mellitus. Methylglyoxal, an aldehyde intermediate compound which is mostly generated from glycation, lipolysis and amino acid oxidation is shown to be elevated in diabetic patients and is believed to be a major precursor for numerous advanced glycation end products (AGEs). Among the diabetic complications is endothelial dysfunction. In the present study we investigated the effect of a clinically relevant concentration of methylglyoxal on vascular function in rat aorta.Methods:Wistar rats were euthanized with CO2 and the aorta was isolated and cut into approximately 3mm wide rings. The aorta ring was then suspended in an organ bath and equilibrated for 90 minutes. After equilibration, tissues were incubated for 15 minutes with L-NAME 100µM or for 40 minutes with 1µM TRPV4 antagonist; HC-067047, followed by methylglyoxal (MGO); 100µM, incubation for 120 minutes. Then the ability of norepinephrine, 3E-7M to produce sustained increases in tension was assessed.Results:Methylglyoxal showed significant relaxation of 3E-7M norepinephrine contraction compared to control (n=11; 97.1±2.2 % relaxation, p < 0.001) after 30 minutes. After pre-incubation with 100µM L-NAME, (n=8; 37±14% relaxation, p ? 0.05) or TRPV4 antagonist (1µM) (n=6; 9±20% relaxation, p?0.05), methylglyoxal failed to induce significant relaxation of the norepinephrine contraction after 30 minutes. Discussion: Methylglyoxal incubation abolished the plateau phase of norepinephrine-induced contraction. This effect was blocked with L-NAME and TRPV4 antagonist, consistent with the idea that methylglyoxal can cause vascular relaxation by activating NOS and/or

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TRPV4 channels. Thus, methylglyoxal produces complex effects on rat aorta at a

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concentration that is seen in human diabetic plasma. These effects are at lower

A Gq – Ca2+ axis controls circuit-level encoding of circadian time in the suprachiasmatic nucleus by recruiting VIPergic intrinsic connectivity Presenting author: Dr Marco Brancaccio ([email protected]) Authors: Marco Brancaccio 1, Elizabeth S. Maywood 1, Johanna E. Chesham 1, Andrew S. I. Loudon 2, Michael H. Hastings 1 Authors' affiliations: Division of Neurobiology, MRC Laboratory of Molecular Biology, Cambridge, CB2 OQH, 2 Faculty of Life Sciences, University of Manchester, Manchester

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DREADDs, neural circuits The role of intracellular transcriptional/post-translational feedback loops (TTFL) within the circadian pacemaker of the suprachiasmatic nucleus (SCN) is well established. In contrast, the role of intercellular signalling and cytosolic rhythms in mediating intercellular control of SCN circadian pacemaking is still poorly understood. We therefore combined viral transduction of SCN slices with fluorescence/bioluminescence imaging to visualize GCaMP3-reported circadian oscillations of intracellular calcium [Ca2+]i alongside activation of Ca2+/cAMP-responsive elements. We phase-mapped them to the TTFL, in circadian time and SCN space, and demonstrated their dependence upon G-coupled vasoactive intestinal peptide (VIP) signaling. We then tested the relative contributions of different G coupled receptor pathways (GPCR) by using a pharmacogenetic approach. Selective GPCR manipulations revealed the individual contributions of Gq, Gs- and Gi to both cytosolic and TTFL circadian rhythms. Remarkably, activation of Gq-dependent (but not Gs- or Gi) pathways in a minority of neurons irreversibly lengthened the period and reduced the amplitude of both cytosolic and TTFL oscillations by specific re-programming of [Ca2+]i and TTFL rhythms across the entire SCN circuit. This re-programing was mediated by intrinsic VIP-ergic signaling, thus revealing a Gq/[Ca2+]i-VIP axis relaying circuit encoding of circadian time to the intracellular TTFL in the SCN.

A neural circuit switch reroutes pheromone signals in male and female brains Presenting author: Mr Johannes Kohl ([email protected]) Authors: Kohl J 1, Ostrovsky AD 1, Frechter S 1, Jefferis GSXE 1 Authors' affiliations: 1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, CB2 0QH Keywords: neural circuits, pheromones, sexual dimorphism, olfaction In Drosophila the male sex pheromone cis-vaccenyl acetate (cVA) elicits different innate behaviours in males and females. First and second order olfactory neurons show identical pheromone responses suggesting that sex genes rewire circuits deeper in the brain. We now use in vivo whole-cell electrophysiology to show that two clusters of third order olfactory neurons have dimorphic pheromone responses. One cluster responds in females, the other in males. These clusters are present in both sexes and share a common input pathway, but sex-specific wiring reroutes pheromone information. Investigating the genetic logic of this switch, we find that the fruitless transcription factor both connects the male-responsive cluster and disconnects the female-responsive cluster from pheromone input. Furthermore selective masculinisation of third order neurons can transform both their morphology and pheromone responses, demonstrating that circuits can be functionally rewired by the cell-autonomous action of a switch gene. http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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This bidirectional circuit switch provides a simple mechanism to activate different behaviours in males and females.

A new disorder of neurotransmission associated with SYT1 (Synaptotagmin-1) mutation Presenting author: Dr Kate Baker ([email protected]) Authors: Kate Baker a, Margriet van Kogelenberg b, Detelina Grozeva a, Nicola Roberts a, Mike Pike c, Ed Blair d, Matt Hurles b, W Kling Chong e, Torsten Baldeweg f, Manju Kurian g, Stewart Boyd g, UK10K, Lucy Raymond a Authors' affiliations: a. Department of Medical Genetics, University of Cambridge, b Wellcome Trust Sanger Institute, Hinxton, Cambridge, c Department of Paediatric Neurology, Oxford University Hospitals NHS Trust, d Department of Clinical Genetics, Oxford University Hospitals NHS Trust, e Department of Radiology, Great Ormond Street Hospital for Children NHS Foundation Trust, f Developmental Cognitive Neuroscience Unit, UCL Institute of Child Health, g Neurosciences Unit, UCL Institute of Child Health Keywords: Neurocognition, New Technologies, genetics, neurodevelopment SYT1 is the essential Ca2+-sensitive regulator of fast, synchronous vesicle exocytosis at excitatory synapses. We present the first known case of a human disorder associated with mutation in SYT1. The clinical features are severe, progressive dyskinetic movement disorder and profound cognitive impairment. Epileptic seizures have not been observed and there are no structural brain abnormalities on clinical MRI. The proband demonstrates a highly unusual electrophysiological profile: abnormal paired-pulse depression of visual evoked potentials, immature auditory evoked potentials and extensive low-frequency oscillatory activity. These features point toward abnormal short-term plasticity, impaired maturation of cortical processing and disturbed development of functional brain networks. Whole exome sequence analysis of DNA from the affected individual and both parents revealed a de novo missense mutation (I368T) at a functionally-critical residue in Synaptotagmin-1 (SYT1). Heterozygous mutation at the equivalent residue in Drosophila blocks activity-stimulated vesicle release resulting in lethal motor impairment (Paddock et al 2011), whilst Syt1 knockdown in mouse alters the dynamics of high-frequency neurotransmission leading to abnormal spectral characteristics and impaired prefrontaldependent learning (Xu et al 2012). This case demonstrates that SYT1-mediated regulation of exocytosis is necessary for motor control and for emergence of higher cognition. Activity-dependent regulation of vesicle release may be a critical mechanism underlying other cases of idiopathic neurodevelopmental disorders.

ALG2 – a new gene that causes congenital myasthenic syndromes Presenting author: Dr judith cossins ([email protected]) Authors: Cossins J 1, Finlayson S 1,2, Carboni N 3, Maxwell S 1, WGS500 consortium 4, McGowan SJ 5, Beeson D 1 Authors' affiliations: 1 Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, UK, 2. John Radcliffe Hospital, Oxford , UK, 3. Department of Public Health, Clinical and Molecular Medicine, Sardinia, Italy, 4. The Wellcome Trust Centre for Human Genetics, Oxford, UK, 5. Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Keywords: Neuromuscular junction, congenital myasthenic syndromes Congenital myasthenic syndromes (CMS) are a group of rare inherited conditions in which neuromuscular transmission is impaired, causing fatiguable muscle weakness. We carried out whole genome sequencing to identify the underlying defect in a patient from a consanguineous marriage who had no mutations in any of the genes known to cause CMS. The patient had an inherited limb-girdle phenotype with decrement and jitter on electromyography, and responded well to pyridostigmine. A homozygous variant, c.203T>G (p.Val68Gly), was identified in ALG2 which co-segregates within the family with an autosomal recessive mode of inheritance. ALG2 encodes ALG2, an enzyme that catalyses early steps in the N-glycosylation pathway. We show that ALG2 expression is enriched at the neuromuscular junction, indicating an important function at this synapse. http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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There was a marked reduction of ALG2 protein expression in a muscle biopsy from the patient compared with control muscle biopsies. The variant also caused a reduction ~30% of control in expression of recombinant protein when expressed in HEK293 cells. Thus the variant is likely to be pathogenic by reducing expression of the protein. Our data indicate that mutations in genes involved in the N-glycosylation pathway may cause a multisystems disorder typical of congenital disorders of glycosylation (CDG), or can produce a relatively mild phenotype that is limited to muscle weakness. They confirm an important role for N-glycosylation for correct functioning of the neuromuscular junction. In addition, muscle weakness found in patients with CDG might be improved with the use of acetylcholinesterase inhibitors.

Altered expression of acid-sensitive two-pore domain potassium (K2P) channels in cancer Presenting author: Miss Sarah Williams ([email protected]) Authors: Williams S 1, Bateman A 2, O'Kelly I 3 Authors' affiliations: Human Development and Health, Faculty of Medicine, University of Southampton 1. Cancer Sciences, Faculty of Medicine, University of Southampton 2. Human Development and Health, Faculty of Medicine, University of Southampton 3. Keywords: Ion Channels, Cancer, Oncomine Potassium channels play fundamental roles in cell behaviours linked to cancer progression. The two-pore domain (K2P) potassium channels are of particular interest as they are active at resting membrane potentials and hence can impact cellular maintenance and proliferative roles. Already K2P2.1, K2P3.1, K2P9.1 and K2P5.1 are reported to show altered expression and/or a functional role in cancer. This study utilises the microarray database Oncomine (www.oncomine.org) to evaluate alterations in mRNA expression of each of the 15 K2P channels in 20 cancers. Expression of acid-sensitive K2P channels (K2P3.1 and K2P9.1) was then further examined within a range of patient carcinoma samples and cancer cell lines. Data from Oncomine reveal that K2P channels show increased expression (compared to matched tissue controls) in a range of cancers including breast, leukaemia and lung with K2P1.1, K2P3.1, K2P5.1, K2P12.1 showing significant increases. Significantly more cancers and channels show decreased expression when compared to controls, with 7 of the 20 cancers examined showing significant underexpression of at least one K2P channel. K2P1.1, K2P3.1, K2P5.1, K2P6.1, K2P7.1, K2P10.1 show wide underexpression across the 20 cancers examined. K2P4.1, K2P16.1, K2P18.1 failed to show any altered expression. K2P3.1 and K2P9.1 were detected at the protein level in > 60 % (n=19) of patient carcinoma samples (lung, oesophageal, pancreatic, renal) examined. RT-PCR and immunofluorescence analysis identified K2P3.1 and K2P9.1 in several cancer cell lines including breast, colon, lung, oesophageal and renal. Whole cell currents of selected cell lines were also examined. We present evidence of altered expression of K2P channels in cancer and a role in cancer cell behaviour. Sensitivity of K2P channels to physiological parameters (pH, pO2 and [glucose]) which are disrupted in the tumour microenvironment supports further investigation within this field.

An animal model of autoimmune epilepsy Presenting author: Dr Sukhvir K Wright ([email protected]) Authors: Wright SK 1, Hashemi K 2, Pettingill P 1, Bartram J 3, Stasiak L 3, Lang B 1, Vincent A 1 and Upton L 3 Authors' affiliations: 1. Nuffield Department of Clinical Neurosciences, University of Oxford. 2. Open Source Instruments Inc., Watertown MA 02472, USA. 3. Department of Physiology, Anatomy and Genetics, University of Oxford. Keywords: Epilepsy, autoantibodies http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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Background /aims Autoantibodies to neuronal surface proteins are found in adult seizure-related disorders such as NMDAR- Ab encephalitis and limbic encephalitis. Our study of new-onset childhood epilepsy has shown that CNS autoantibodies are also found in a proportion of these patients. The aim of this study is to determine whether these antibodies are pathogenic and epileptogenic in-vivo. Method Purified IgG from NMDAR-Ab positive patients and healthy controls was injected ICV into mice implanted with wireless EEG transmitters. In-vivo electrophysiology recordings were used to detect spontaneous and induced epileptiform activity. Seizure susceptibility was also tested 48 hours post-injection. Results Mice injected with NMDAR-Ab positive IgG displayed increased seizure susceptibility and specific staining of human IgG in the hippocampus post-mortem. NMDAR-Ab related EEG abnormalities were noted in preliminary EEG analysis. Future work Passive transfer of NMDAR-Ab positive IgG displays their epileptogenic potential. Future work will investigate the underlying pathogenic mechanism in ex-vivo slices.

Benzamil inhibits neuronal and heterologously expressed SK channels Presenting author: Dr Marisol Sampedro Castaneda ([email protected]) Authors: Sampedro Castaneda M, Tonini R, Pedarzani P, Stocker M Authors' affiliations: Research Dept. of Neuroscience, Physiology and Pharmacology, University College London, London, UK Keywords: Neuropharmacology, Calcium transient, calcium activated potassium channel, hippocampal neuron, afterhyperpolarization Calcium activated K+ channels are expressed in hippocampal neurons, where they participate in the regulation of excitability and in the local feedback modulation of Ca2+ influx, directly influencing neuronal computation. Using selective SK channel inhibitors and enhancers, feedback regulation of Ca2+ influx triggered by back-propagating action potentials through an interplay between SK and L-type Ca2+ channels was recently described in the proximal dendrites of hippocampal neurons. Besides affecting the amplitude of Ca2+ transients, SK channel inhibition also prolonged their duration, but the role of Ca2+ clearance mechanisms and their link to SK channel activity was not established. In this work, we used the Na+/Ca2+ exchanger (NCX) inhibitor benzamil to examine the interaction between Ca2+ extrusion and SK channels in the regulation of dendritic Ca2+ signals. Benzamil increased not only the duration, but also the amplitude of the Fluo-4 signals generated in the proximal apical dendrite by back-propagating action potentials. The unexpected effect of benzamil on the amplitude of the Ca2+ transients, similar to what we had observed with SK channel blockers, suggested this compound might act as a SK channel inhibitor. We tested this hypothesis on both neuronal SK currents and heterologously expressed SK (1-3) channels. Both were inhibited by benzamil, in the case of recombinant SK channels with IC50 ranging from 35 to 67 µM. This corroborates our findings on the modulation of Ca2+ signals by SK channels and points at benzamil and other amiloride analogues as lead compounds for the development of selective and reversible SK channel inhibitors. The results, additionally, prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native systems, given the spectrum of ion channels and exchangers it targets within a similar range of concentrations.

Biphasic control of histamine cells by hypothalamic hypocretin/orexin neurons Presenting author: Dr Cornelia Schöne ([email protected]) Authors: Schöne C 1, Burdakov D Authors' affiliations: Neurophysiology, MRC National Institute for Medical Research Keywords: orexin, histamine, neuropeptide transmission, body homeostasis

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Hypothalamic hypocretin/orexin (OX) neurons help to maintain body homeostasis by regulating wakefulness, feeding, drinking, reward seeking and basic body functions such as blood pressure and heart rate. However, it remains poorly understood how OX cells integrate with other brain areas to specifically modulate different aspects of body homeostasis. Tuberomammillary histamine (TMN HA) neurons are a major projection target of OX cells and are believed to mediate the arousal promoting effects of OX cells. Using acute brain slice preparations, here we selectively stimulated OX fibers in the TMN using optogenetics and recorded postsynaptic responses in HA cells. We found that both glutamate and OX peptides were released from OX fibers and contributed to HA cell depolarisation. Glutamate actions were fast, but were unable to sustain signaling in OX to HA circuits during prolonged stimulations. On the other hand, only prolonged stimulation at high frequencies produced significant OX peptide mediated excitation in HA cells. This showed a slowly rising time course throughout the stimulus duration and long lasting effects for minutes after the stimulation end. These results show that time course and release conditions for OX and glutamate mediated signaling were complementary and that each transmitter alone was unable to propagate OX to HA signaling effectively. It also becomes evident that different transmitters may encode OX signaling to postsynaptic neurons and that this depends on OX activity patterns.

Building and annotating a knowledge repository of synaptic signalling Presenting author: Ms Martina Fröhlich, ([email protected]) Authors: Martina Fröhlich 1, Nicolas Rodriguez 1,2, Junmei Zhu 2, Nicolas Le Novère 1 Authors' affiliations: 1. Babraham Institute, Cambridge, 2. EMBL-EBI, Hinxton Keywords: comprehensive pathway map, data repository, glutamatergic synapse, synaptic signalling Glutamatergic synapses play an important role in learning and memory. Synaptic strength, that is the change in postsynaptic potential after a presynaptic neurotransmitter release, can be varied and is tightly regulated via various intracellular signalling pathways. Within the context of the EU project SynSys, we built a knowledge repository of the signalling processes involved in the postsynaptic part of a glutamatergic synapse. This encompasses graphical representations of relevant signalling pathways at different levels of granularity as well as a database where information can be effectively stored and retrieved. A global coarse map provides an overview of the processes involved in a glutatamergic synapse. It illustrates the crosstalk between pathways and the existence of various feedback and feedforward loops. In addition, detailed maps for calcium, MAP kinase, and phosphoinositide signalling are provided. The collection of maps was generated using a combination of existing knowledge available in public databases such as KEGG and Reactome and was manually curated using scientific literature, the Allen Brain Atlas as well as proteome and interactome data available within SynSys. Both, the maps and the database, are annotated using MIRIAM URIs and link to external sources such as PubMed, ChEBI and UniProt. The maps were build in CellDesigner and are available in SBML and SBGN formats. In conclusion, we provide a uniquely curated, annotated and comprehensive data repository of synaptic signalling which can be directly used for the development and refinement of mathematical models.

Calmodulin inhibits TRPA1 ubiquitination and membrane tra cking induced by mustard oil Presenting author: Mr Raquibul Hasan ([email protected]) Authors: Hasan R, Li L, Zhang X Authors' affiliations: Department of Pharmacology, University of Cambridge, Cambridge, UK Keywords: TRPA1, ubiquitination, membrane trafficking The TRPA1 ion channel is a molecular target of various noxious stimuli and is involved in pain. Mustard oil (MO) has been reported to cause nociception by directly exciting TRPA1 and increasing its surface expression in sensory neurons. However, the underlying mechanisms causing increased TRPA1 trafficking by MO is unknown. We herein investigate whether MO-induced TRPA1 surface trafficking is due to increased TRPA1 http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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ubiquitination by using molecular biology and protein biochemistry techniques. We found that TRPA1 was weakly ubiquitinated under basal condition. The ubiquitination of TRPA1 was dramatically enhanced after stimulation with MO. Interestingly, MO also stimulated a concurrent increase in the membrane expression of TRPA1.We further demonstrated that TRPA1 underwent multiubiquitination instead of polyubiquitination by generating ubiquitin mutants unable to form polyubiquitin chains. The modification of TRPA1 by ubiquitin was prevented in the absence of extracellular Ca2+. However, TRPA1 ubiquitination was not affected by chelating intracellular Ca2+ with BAPTA-AM. Remarkably, overexpression of calmodulin (CaM) completely abolished both basal and MO-induced TRPA1 ubiquitination, and also coincidentally abolished increased TRPA1 membrane expression. Furthermore, TRPA1 was found to bind to CaM in a Ca2+dependent manner. However, calcium-incompetent CaM1234 also abolished TRPA1 ubiquitination, suggesting that such a reduction in TRPA1 ubiquitination is mediated by an indirect interaction of CaM to TRPA1. Our findings reveal a novel mechanism for the regulation of TRPA1 channel trafficking by ubiquitin and CaM. Ubiquitination of TRPA1 following MO stimulation might represent a mechanism underlying TRPA1 trafficking. CaM inhibits TRPA1 trafficking perhaps via inhibition of its ubiquitination

CELLULAR MECHANISMS OF SPIKE TIMING-DEPENDENT LONG-TERM DEPRESSION IN THE HIPPOCAMPUS Presenting author: Miss Paloma Duque-Feria ([email protected]) Authors: Duque-Feria P 1, Andrade-Talavera Y 1, Rodríguez-Moreno A 1 Authors' affiliations: 1. Laboratory of Cellular Neuroscience and Plasticity, University Pablo de Olavide, Seville, Spain Keywords: STDP, Hippocampus, NMDAR Spike timing-dependent plasticity (STDP) is a strong candidate for a synaptic mechanism involved in cortical development and learning and memory. The main objective of this work was to determine the cellular mechanisms of this form of LTD. We conducted experiments in hippocampal slices prepared from P9-P16 mice using the whole-cell configuration of the patch clamp technique. To induce t-LTD, a post-pre pairing protocol (with the presynaptic activity ocurring 18 ms after a postsynaptic action potential) was applied after a stable EPSP baseline period of 10 min. EPSP slope was monitored for at least 30 min after the pairing protocol. We found that a post-before-pre pairing protocol induced robust t-LTD (75 ± 5% of control, n = 9). This t-LTD was completely blocked by the NMDA receptors antagonist DAP5 (50 µM, 118 ± 7%, n = 7). The loading of the postsynaptic neuron with the usedependent non-competitive NMDA receptor antagonist MK-801 failed to prevent the induction of t-LTD (76 ± 4 %, n = 6) indicating that t-LTD induction is mediated by nonpostsynaptic NMDA receptors. To determine the subunit composition of NMDA receptors mediating this form of LTD we performed experiments in the presence of subunitpreferring NMDA receptor antagonists. t-LTD was prevented by PPDA (93 ± 8%, n = 5) but not by NVP-AAM077 (73 ± 6%, n = 6) or Ro 25-6981 (76 ± 5%, n = 5) indicating that requires NMDA receptors containing GluN2C/D subunits but no GluN2A or -2B. Finally, to determine the possible involvement of CB1 receptors in t-LTD, we performed experiments in the presence of the antagonist AM251 (3 µM). In this situation t-LTD induction was completely prevented (104 ± 8%, n = 9). t-LTD induction requires the activation of non-postsynaptic GluN2C/2D subunit–containing NMDA receptors and CB1 receptors.

Common principles and dynamics underlie cortical face perception and insect swarm intelligence Presenting author: Dr Jonathan O'Keeffe ([email protected]) http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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Authors: O'Keeffe Jonathan 1, Kriegeskorte Nikolaus 2 Authors' affiliations: MRC Cognition and Brainsciences Unit, Cambridge Keywords: Neurocognition, Computational, Neuroscience, Psychophysics The human neocortex is often cited as a structure which differentiates homo sapiens from the rest of the animal kingdom. Of the functions supported by the neocortex face perception is a strong candidate for a primate-specific, perhaps even species-specific, function. Here we present evidence from psychophysical experiments in humans and a neuronal modelling study that suggest that the process of evidence accumulation in face perception shares its dynamics, and possibly computational and algorithmic rationales (in Marr's sense), with recently elucidated decision making processes implemented by swarming honey-bees when choosing a new nest site. Our results suggest that neurons in human cortical face representations may collectively implement a similar dynamic decision process as bees, involving mutual inhibition and a multistable drift-diffusion process. Comparing two functionally remote tasks in phylogenetically remote species reveals common principles of biological intelligence that may underlie insect swarm intelligence and certain aspects of human cognition.

Computational Optogenetic Models for Controlling Neural Circuits with Light Presenting author: Dr Konstantin Nikolic ([email protected]) Authors: Jarvis S 1, Grossman N 2, Schultz SR 2 and Nikolic K 3 Authors' affiliations: 1. Dept of Bioengineering, Imperial College London, 2. Dept of Biological Eng and Brain and Cognitive Sciences, MIT, 3. Dept. of Electrical & Electronic Eng, Imperial College London Keywords: New Technologies, optogenetics, computational neurscience Optogenetics is a technology that combines genetic and optical techniques in order to induce light sensitivity of excitable cells. It allows very precise targeting and fast modulation of some biological functions. The field of optogenetics has the potential to be one of the most important new techniques in neuroscience because it offers the best promise for studying functional circuit connectivity in experimental neuroscience and has direct implications in future therapeutic strategies. Here we present a computational modeling technique for two classical mechanisms for cells optical activation and silencing: channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR). The technique was demonstrated on an example of the neural response of a layer 5 cortical pyramidal neuron (L5) with different areas of illumination: whole cell, apical dendritic tree, somatic and axon initial segment (AIS) illumination. We show that whole-cell illumination of NpHR most effectively hyperpolarizes the neuron and is able to silence the cell even when driving input is present. However, when ChR2 and NpHR are concurrently active, the relative location of each illumination determines whether the response is modulated with a balance towards depolarization. The methodology developed in this study will be significant to interpret and design optogenetic experiments and in the field of neuroengineering in general.

Cross-isoform promiscuity of potent, state-dependent and selective inhibitors of human voltage-gated sodium channels Presenting author: Dr Srinivasan Kanumilli ([email protected]) Authors: Srinivasan Kanumilli1, Tim Dale1, John Rauch2, Kevin Allard2, Libby Oupicka2, Dave Rock2, Vince Groppi2 & Derek Trezise1 Authors' affiliations: 1Essen Bioscience Ltd, BioPark, Broadwater Road, Welwyn Garden City, United Kingdom, 2Essen Bioscience Inc, 300 West Morgan Road, Ann Arbor, Michigan, USA. Keywords: Neuropharmacology, Neurocognition, Affective Disorders, Neurodegenerative Disorders, voltage gated sodium channel, neuropharmacology, drug discovery, ion channel, channelopathy Submitted by email. http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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Cux1 regulates long-range layer II-III inter-hemispheric connections by modulating AIS function Presenting author: Mrs Maria Fernanda Rodríguez Tornos ([email protected]) Authors: 1Fernanda M. Rodríguez-Tornos, 2José Antonio Esteban and 1Marta Nieto Authors' affiliations: Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus de Cantoblanco, Madrid 28049, Spain, 2Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, 28049 Spain Keywords: Neurocognition, Neuropsychiatry - Autism, layer II-III, cortex, connectivity, ais, potassium channel, excitability, autism The development of the corpus callosum (CC) is essential for cerebral cortex functions. Dysgenesis of the CC is one of the few clinical features that correlate with autism spectrum disorders (ASD). This contributes to reduced long-distance connectivity, whereas local circuits are thought to be less affected. We show that Cux1 loss of function in layer II-III CC neurons abolished contralateral connectivity, but not midline crossing or ipsilateral innervation. Cux1 regulates the levels of Kv1.1 and Kv1.3, which clusters at the axonal initial segment (AIS). In vivo rescue experiments demonstrated that innervation defects are due to reduced Kv currents, higher excitability and abnormal plasticity of the AIS, whereas they are independent of somatic firing. By modulating Kv1 channels and AIS function, Cux1 thus controls efficacy of axon action potentials and long-range axonal stabilization. This mechanism has important implications for understanding the biology of developmental cognitive disorders, in particular ASD.

Demyelinated axons regulate their own remyelination via glutamate signalling to oligodendrocyte precursor cells Presenting author: Dr Helene O B Gautier ([email protected]) Authors: Gautier H., Evans K., Lundgaard I., Lao-Peregrín C., Franklin R.J.M., Káradóttir R.T. Authors' affiliations: Wellcome Trust/MRC Cambridge Stem Cell Institute & Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom Keywords: Neuroregeneration, Glutamate, Oligodendrocyte precursor cells, remyelination During development, some OPCs differentiate into myelinating oligodendrocytes while others remain into adult life, where they comprise approximatley 5% of CNS cells. In demyelinating disease such as multiple sclerosis, adult OPCs differentiate into new oligodendrocytes that remyelinate the axons, although this process eventually fails, leading to sustained clinical deficits. Glutamate and electrical activity influence OPC differentiation and myelination in normal development. OPCs receive synaptic input from unmyelinated axons. Both OPCs and mature oligodendrocytes respond to glutamate via AMPA and NMDA receptors. Here we examine the role of glutamate signalling in remyelination following experimental demyelination. We voltage-clamped OPCs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by NG2immunolabelling. Recruited OPCs mainly expressed AMPA receptors at the peak of the OPCs proliferation, as over 90% of the glutamate evoked current was blocked with NBQX (AMPA/Kainate receptor antagonist) and unaffected by AP5 (NMDA receptor antagonist). The demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development. Critically, the demyelinated axons established synapses with OPCs expressing voltage-gated sodium currents. Pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation. These results indicate that 1) glutamate currents are mainly mediated via AMPA and kainate receptors during the OPC recruitment 2) OPCs establish synaptic contact with demyelinated axons and 3) the neuronal activity is essential for remyelination. Together, these data suggest that demyelinated axons remain active and communicate with OPCs through glutamate release during the regenerative process to guide their remyelination.

http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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Developmental characterization of human pluripotent stem cell di erentiation to nociceptors Presenting author: Dr KAUSALIA VIJAYARAGAVAN ([email protected]) Authors: Iceta R 1 , Bossé M 1, Bruzos-Cidon C 2, Ugedo L 2, Ferrer-Montiel A 3 and Vijayaragavan K 1 Authors' affiliations: 1. Neurodegenerative Disease Program, Inbiomed Foundation; 2.Dept. Pharmacology, University of the Basque Country; 3. Inst. Molecular Biology y Cellular, Universidad Miguel Hernández. Keywords: Neuroregeneration, Human Pluripotent Stem Cells, Nociceptors, BRN3a, SOX2, Nav1.7 & Nav1.8 Human embryonic stem cells (hESCs) and the induced pluripotent stem cells (hiPSCs), collectively known as human pluripotent stem cells (hPSCs), represent an invaluable opportunity to dissect complex cellular and molecular events that occur during early nociceptor development. Importantly the differentiation process from hPSCs allow the unique means to track and isolate intermediate cells (precursors) that only transiently exist in vivo like the neural crest cells and progenitor cells for the peripheral nervous system. While specification of peripheral sensory neurons has been studied in animal models, investigation into the embryonic determinants for the emergence of human nociceptive sensory neurons remains elusive. Here we present a study describing the emergence of sensory neurons from hPSCs adapted under serum and feeder free conditions. Early during differentiation (day 0), neuroepithelial cells derived from hPSCs stained positive for p75, HNK1, A2B5 and MAP5. The neuroepithelial cells were also positive for BRN3a, however were localized in the perinuclear region as opposed to discreet punctuated pattern later during the differentiation (days 25-30). We also observed two waves of SOX2 expression peaking at days 0-4 and days 18-30. Interestingly at day18, we observed a mutually exclusive localisation of high SOX2+ and high BRN3a+ neurons. By day 30, the neurons were positive for peripherin, TRKA, Nav1.7 and Nav1.8 channels. TRKA+ neurons were positive for Nav1.7 and Nav1.8, however Nav1.7 staining in the soma was co-localised with TRKA, while this arrangement was not observed along the axon. As for Nav1.8 positive neurons, TRKA did not co-localise with the TTX-R channel neither in the soma nor the axon. Whole cell recording revealed two types of Na currents, a fast gating and voltage dependence component showing TTX sensitivity or typical slow gating Na currents with persistent properties.

Di erential responses to emotional stimuli in OCD and Social Anxiety Disorder Presenting author: Dr Annette B Bruhl ([email protected]) Authors: Brühl AB 1,2, Weidt S 3, Lutz J 2, Rufer M 3, Herwig U 2, Sahakian BJ 1, Robbins TW 1 Authors' affiliations: 1 Behavioural and Clinical Neuroscience Institute, University of Cambridge, 2 Department of Psychiatry, Psychotherapy, and Psychosomatics, University Hospital of Psychiatry Zurich, 3 Department of Psychiatry and Psychotherapy, University Hospital Zurich Keywords: Affective Disorders, fMRI, OCD, Social Anxiety Disorder, Emotion The classification of obsessive-compulsive disorder (OCD) as an anxiety disorder is the subject of a long-standing debate. To investigate underlying basic emotion processing circuits we compared patients with OCD and with Social Anxiety Disorder (SAD) during the perception of non-specific emotional stimuli using functional magnetic resonance imaging. Each group comprised 16 patients. During the perception of neither OCD- nor SAD-specific, negative emotional pictures (compared to neutral pictures), patients with OCD showed stronger activations than SAD in left middle insula and left fusiform gyrus, whereas patients with SAD activated more strongly the posterior cingulate, left precuneus and left angular gyrus. During the perception of positive emotional pictures (contrasted against neutral pictures), patients with OCD showed stronger activations in the left middle/inferior frontal gyrus, whereas patients with SAD had stronger activations in the mediodorsal thalamus, right dorsal superior temporal gyrus and posterior cingulate. In http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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summary, these results revealed no significant differences at the level of amygdala and anterior insula, structures known to be involved in the processing of emotional stimuli. However, there are differences between the two diagnostic groups at the level of perceptual processing and attentional brain circuits. Further research within broader diagnostic categories might help to understand the relative roles of common and differential neural circuits in these disorders.

Dissociation of the Kv1.1/Kvb1 channel complex with a small molecule inhibits channel inactivation and reduces neuronal excitability Presenting author: Dr Christian Wolff ([email protected]) Authors: Wolff C, Mullier B, Ghisdal P, Niespodziany I, Montel F, Cornelis S, André V, Burton M, Miller K Authors' affiliations: UCB Pharma, Neuroscience Research, Braine l’Alleud, Belgium Keywords: Neuropharmacology, Kv1.1, epilepsy, HTS, Electrophysiology Kv1.1 (KCNA1) channels are expressed in excitatory and inhibitory neurons where they contribute to the control of action potential firing and neurotransmitter release. Kv1.1 channels are characterized by rapidly inactivating A-type currents resulting from the specific association of the cytoplasmic Kvb1 subunit. The deletion of Kv1.1 channels in mice translates into ataxia and spontaneous seizure activity and genetic mutations of KCNA1 have been associated with episodic ataxia type 1 (EA1) and seizure activity in humans. Recent studies demonstrated that the lentiviral over-expression of Kv1.1 in epileptic mice leads to reduced seizure activity. We hypothesized that modulating Kv1.1 activity, by inhibiting Kv1.1 inactivation with a small molecule, would lead to decreased neuronal excitability and anticonvulsant activity. We developed a high throughput screening assay to identify small molecules able to dissociate the complex formed by the Kv1.1-T1 domain and the Kvb1 proteins. The tagged protein domains were expressed in HEK cells and formation of the protein complex was measured by the ALPHAscreen technology. We screened 137,000 compounds and identified several series of molecules able to disrupt the protein complex in the micromolar range. Selected compounds were tested by TEVC on Kv1.1/Kvb1 channels expressed in Xenopus oocytes and were shown to inhibit the inactivation process. We further evaluated a selected compound on intrinsic neuronal excitability using current clamp recordings in granule cells of the dentate gyrus. This compound, tested at 100µM, significantly reduced by 80% the number of action potentials fired during a single train. This study described for the first time a rationale drug discovery approach for the identification of novel ligands that inhibit Kv1.1 channel inactivation and provides pharmacological evidence that such a mechanism translates into physiological effects by reducing neuronal hyper-excitability.

Dopamine release is regulated by N-, Q-, T- and L-type voltage dependent calcium channels (VDCCs) in dorsal striatum but only N- and Q-types in ventral striatum Presenting author: Ms Katherine Brimblecombe ([email protected]) Authors: Brimblecombe K, Cragg S Authors' affiliations: Department of Physiology, Anatomy and Genetics, University of Oxford Keywords: Neuropharmacology, Neurodegenerative Disorders, Dopamine, voltage-gated calcium channel, Parkinson's disease, alpha synuclein, electrochemistry Midbrain dopamine (DA) neurons in the substantia nigra pars compacta (SNc) that project to caudate putamen (CPu) show greater sensitivity to Parkinson’s disease (PD) than those in the ventral tegmental area (VTA) that project to nucleus accumbens core (NAc). These two neuron types are known to differ in in their roles for L-type voltage-dependent Ca2+ channels (VDCCs) in action potential generation. However the roles of VDCCs in the large axonal fields have been incompletely defined. Work to date has been confounded by VDCCs operating on striatal cholinergic interneurons (ChIs), which powerfully regulate DA release. Here we have investigated the roles of VDCCs in the control of axonal DA release in both CPu and NAc, in the presence of the nicotinic ACh receptor (nAChR) antagonist http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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Dihydro-?-erythrodine (Dh?E) to remove the confounding effects of striatal ChI. We assessed the role of the different VDCCs in controlling striatal DA release using fast scan cyclic voltammetry and specific blockers of the N, P/Q, L, and T-type channels (?Conotoxin GVIA(100 nM), ?-Agatoxin IVA (200 nM), Isradipine(5 ?M) and NNC 55-0396 (1 ?M) respectively in acute coronal mouse brain slices. We find DA release in CPu to be more sensitive to VDCC inhibition than NAc. We find that DA release is controlled by N>P/Q>T> L types in CPu, but only N andP/Q-types in NAc . L-type VDCCs appear to control DA release in an ?-synuclein dependent manner, these findings may provide key insights for PD.

EphB1/Ephrin-B1 reverse signalling induces astrocyte activation Presenting author: Miss Giulia Tyzack ([email protected]) Authors: Giulia Tyzack, Tomasz Cymes, Nike Lau and Andras Lakatos Authors' affiliations: John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Keywords: Neuroregeneration, astrocyte reaction, synaptic plasticity, ephrin signalling Emerging evidence suggests that signalling by damaged neurones leads to reactive astrocyte response that may have a fundamental impact on synaptic reorganisation. Soluble factors, such as cytokines,have been described to induce such astrocyte activation. However, rapid contact dependent signalling between damaged neurones and astrocytes is also a possibility. In light of recent findings of EphB1 upregulation on injured neurons closely surrounded by reactive astrocytes, we hypothesized that EphB1 can signal to ephrin-B1 expressing astrocytes. To test this in simplified in vitro assays, purified astrocyte cultures were treated with clustered-EphB1-Fc, and the characteristic features of glial activation, such as cytoskeletal protein expression, STAT3 phosphorylation, and p-STAT3 nuclear transfer were monitored. We have demonstrated that 1) Astrocytes express ephrin-B1, 2) EphB1 triggers a two-fold increase in GFAP and ezrin expression, 3) EphB1 induces both STAT3 phosphorylation and nuclear transfer by a three-fold value, and 4) all effects were significantly diminished by knocking down ephrinB1 in astrocytes.Our data show that EphB1 induces astrocyte activation by triggering ephrinB1-mediated reverse signalling via STAT3 activation. This work therefore suggests a potential novel route in neuron-astrocyte communication after injury. A further characterisation of this signalling pathway may provide a better understanding of mechanisms underlying glial activation and its role in plasticity.

Explaining the regulatory e ect of monovalent ions on kainate receptors Presenting author: Dr Maria Musgaard ([email protected]) Authors: Maria Musgaard 1, G. Brent Dawe 2, Bryan A. Daniels 2, Mark R.P. Aurousseau 2, Derek Bowie 2 and Philip C. Biggin 1 Authors' affiliations: 1 Department of Biochemistry, University of Oxford, Oxford, UK; 2 Department of Pharmacology & Therapeutics, McGill University, Montreal, QC, Canada Keywords: Ligand-gated ion channels, glutamate receptors Ligand-gated ion channels activated by glutamate binding, ionotropic glutamate receptor (iGluRs), play crucial roles in the CNS, e.g. in learning and memory. Furthermore, iGluRs are implicated in many CNS disorders. Kainate receptors (KARs) belong to the iGluR family and are distinct from other family members as they require binding of monovalent extracellular ions (Na+ and Cl.) in addition to agonist for activation. The agonist binds to the extracellular ligand binding domain (LBD), triggering opening of the transmembrane cation-selective channel. The overall iGluR structure, determined for the similar AMPA receptor, shows that the extracellular domains of the tetrameric receptor are organised as a dimer of dimers, whereas the channel domain has four fold symmetry. Binding sites for the regulatory ions have been identified at the KAR LBD dimer interface. Despite knowledge of the binding sites, the precise roles of the ions in activation and desensitisation are unclear. To explain the requirement for these ions, we have studied the effect of different mutations at the LBD interface region, including two cross-linking mutations, by atomistic molecular dynamics simulations combined with http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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electrophysiological single-channel recordings. Our simulation data explain structurally why the two cross-linking mutants in electrophysiology surprisingly showed very different channel conductance and underline the importance of a tightly packed LBD dimer interface for normal channel activity, stabilised by ion binding. Thus, the extracellular ions are required to provide maximal efficiency of opening upon agonist binding. Moreover, our results suggest that occupancy of the cation pocket determines the onset of receptor desensitisation. If the agonist unbinds from the open state before cations, the receptor returns to the inactive state, whereas if the cations unbind first, the receptor enters the desensitised state. Hence, KAR activity is strongly regulated by extracellular ions.

Exploring astrocyte response to neuronal signals in axon damage by genetically encoded calcium indicators: a platform for optimising astrocyte mediated plasticity Presenting author: Mr Daniel Barson ([email protected]) Authors: Daniel Barson and Andras Lakatos Authors' affiliations: John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Keywords: New Technologies, Neuroregeneration, Ca imaging, astrocyte reaction, synaptic plasticity, axon damage The role and mechanism of grey matter astrocyte response to distant neuronal injury remains poorly understood. Previous findings by our laboratory have demonstrated that remotely activated astrocytes promote structural synaptic plasticity (Tyzack et al., 2013). To further explore the pathways involved in neuron-astrocyte signalling in response to neuronal cues in CNS damage, we developed a novel method to measure calcium flux in astrocyte networks in organotypic entorhino-hippocampal slice cultures. Specifically, we use an adeno-associated virus containing the genetically encoded calcium indicator GCaMP6s within a double-floxed cassette to achieve robust expression of the indicator exclusively in astrocytes. Using real time two-photon and conventional microscopy, this system allowed us to reliably measure changes in astrocyte calcium dynamics at different time points after distant perforant pathway lesioning or treatments with signaling molecules. In a transgenic system, we also compared the effects of normal to diminished astrocyte reactivity on network calcium waves with a view to assess how it influences synaptic activity. This model may help us understand how glial cell responses can be optimised to facilitate plasticity and functional recovery after neuronal damage.

Glutamate and GABA receptors in Fragile X Syndrome (FXS) Presenting author: Miss Jantine Broek ([email protected]) Authors: Broek J.A.C., Guest P.C., Rahmoune H., Bahn S. Authors' affiliations: Dept. of Chemical Engineering and Biotechnology Keywords: Neuropharmacology, Neuropsychiatry - Autism, Fragile X Syndrome, Autism. Glutamate, GABA, Mass Spectrometry Fragile X syndrome (FXS) is a single gene disorder that is the most common heritable cause of intellectual disability and is associated with autism in 20-30% of the cases. To achieve a better understanding of FXS, the disorder should be investigated at the molecular level to establish a unique signatures. FXS is caused by a trinucleotide repeat in the Fmr1 gene, which leads to silencing of the FMRP protein that regulates the translation of specific proteins. Determining the identity and function of these proteins might lead to the underlying cause of FXS and autistic behaviour. Using mass spectrometry-based molecular characterization, proteins that are affected in Fmr1 KO mice models were identified. This study shows that the proteins that are significant different in Fmr1 KO compared to control mice are associated with the mGluR theory of FXS. This theory describes the influence of FMRP on long term depression (LTD) activated by the metabotropic glutamate receptors (mGluRs). In individuals with FXS, inhibition of LTD is absent and stimulation of mGluR and consequent internalization of AMPA receptors leads to increased LTD. In addition to mGluR theory associated findings, GABA associated http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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proteins are also found to be differentially expressed. GABA involvement in FXS is a not yet reported feature and justifies further investigation. These results implicate glutamate and GABA signalling in pathophysiology of FXS.

Glycine receptor antibodies: Clinical observations and serological ndings Presenting author: Dr Alexander Carvajal ([email protected]) Authors: Maria Isabel Leite, Linda Clover, Mark Woodhall, Patrick Waters, Angela Vincent Authors' affiliations: Nuffield Department of Clinical Neurosciences, University of Oxford Keywords: Neuroimmunology, Glycine receptor, antibody, Progressive encephalomyelitis with rigidity and myoclonus (PERM) Progressive encephalomyelitis with rigidity and myoclonus (PERM) is characterized by muscular rigidity, stimulus-sensitive spasms plus brainstem symptoms and may have an autoimmune aetiology since recently PERM patients have been shown to have glycine receptor (GlyR?1) antibodies and respond to immunotherapies. However. the clinical phenotype may be more extensive. We studied the clinical features of patients with GlyR antibodies and examined the sera using cell-based assays, immunohistochemistry and cultured neurons. Serum samples from 42 patients sent for routine antibody diagnostic testing bound to the alpha 1 subunit of the GlyR?1 mainly with titres between 1:20 and 1:60,000, but also cross-reacted with other alpha subunits. Spasms, stiffness, excessive startle and touch sensitive myoclonus were the most common symptoms but brainstem disturbances, cognitive deficits with encephalopathy/seizures and autonomic signs were also found; 4 patients had tumors. Most patients were highly disabled but responded well to immunotherapy, but 3 patients died unexpectedly. On rat tissue the patients’ serum IgG bound to cell bodies and to the neuropil of the brainstem and the spinal cord, where they co-localized with monoclonal antibodies to GlyR?1. However, some sera also bound to other brain regions sometimes without co-localization with monoclonal antibodies to GlyR?1. Some of these sera contained GAD antibodies in addition to GlyR. Some sera immunolabelled hippocampal cultured neurons which don’t express GlyR?1, suggesting the presence of antibodies to other antigens or the binding of these sera to other GlyR alpha subunits. Patients with GlyR antibodies have a wide clinical presentation; in addition to the motor symptoms some patients also exhibit brainstem and encephalopathic disturbance. Most patients responded well to immunotherapies. The GlyR antibodies bind to multiple regions in the CNS, which could explain the multifocal clinical features present in these patients.

Integration of automated patch clamp systems and logistic models in the CardiochannelGram (CCG) for better prediction of cardiac risk Presenting author: Mr Hiten Patel ([email protected]) Authors: Kramer JK 1, Myatt GJ 2, Obejero-Paz C 1, Bruening-Wright A 1, Kuryshev YA, Brown AM Authors' affiliations: 1. ChanTest Corp, Cleveland, OH, USA, 2.Leadscope Inc, Columbus, OH, USA Keywords: Affective Disorders, cardiac safety Drug-induced inhibition of the cardiac hERG potassium channel is recommended by ICH and FDA to predict delayed cardiac repolarization (DR) and cardiac risk. The consequent QTc prolongation is a surrogate marker of Torsade de Pointes (TdP), a rare but potentially lethal iatrogenic outcome. Drugs with effective therapeutic plasma concentrations (ETPC) within 30-fold of their hERG IC50s are thought to be dangerous despite the fact that multiple ion channel effects (MICE) can mitigate DR. Here we demonstrate that logistic regression models, which integrate MICE, predict TdP with much greater certainty than the hERG safety ratio (hERG IC50/ETPC or safety margin (SM)) alone. To this end we measured hERG, Nav1.5, Cav1.2, Kir2.1 and KvLQT1/mink IC50 values of 55 drugs (32 + TdP and 23 –TdP) from multiple classes using automated patch clamp http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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systems including Qpatch and PatchXpress. The sensitivity of the automated patch clamp platforms was evaluated in a comparison to manual patch clamp. ETPC values and the torsadogenic liability of drugs was obtained from the literature, package inserts and Arizona CERT. Logistic regression models were constructed; one using the hERG SM alone, the others integrating hERG SM, Nav1.5 SM and/or Cav1.2 SM data. The predictive power of each model was evaluated using the likelihood ratio test. Leave-one-out cross validations were performed and each model’s accuracy was determined by comparing receiver-operating characteristics (ROC, sensitivity vs. 1specificity). Models that include Nav1.5, Cav1.2 or both variables are statistically significant better predictors of TdP liability than the model that contains only hERG (Model 1). Model 1 had a ROC area under the curve (AUC) of 0.77 and Model 2, that includes hERG, Nav and Cav1.2 SMs, significantly improved accuracy showing a ROC AUC of 0.91. Thus, Model 2 that incorporates the concept of MICE in the CardioChannelGram ™ (CCG™) is a robust nonclinical predictor of cardiac risk.

Investigating mitochondria in Down's syndrome Presenting author: Ms Kate McAllister ([email protected]) Authors: McAllister CJ 1, Sleigh A 2, Watson L 3, Walpert MJ 1, Zaman SH 1, Holland AJ 1 Authors' affiliations: 1. Dept Psychiatry, University of Cambridge, 2. Wolfson Brain Imaging Centre, University of Cambridge, 3. Wellcome Trust Clinical Research Facility, Cambridge University Hospitals Keywords: Neurodegenerative Disorders, Down's syndrome, mitochondria, intellectual disability, ageing Down’s syndrome (DS) is a disorder of intellectual disability (ID) and affects around 1 in 700-1000 live births. DS is caused by the triplication of all or most of chromosome 21. Many characteristics of DS including but not limited to weak muscle, exercise intolerance, and impaired sensory functioning are also signs that might also raise suspicion for a primary mitochondrial disease. DS is associated with precocious aging and is also a major risk factor for Alzheimer's disease (AD) due to triplication of the amyloid precursor protein gene, with the majority of people with DS demonstrating AD neuropathology by the fifth decade. Mitochondrial dysfunction and mitochondrial DNA mutations have been noted in AD and are outlined as a hallmark of ageing in the general population, which suggests that defective energetics could be a modifying factor in precocious ageing as well as Alzheimer’s disease in Down’s syndrome (DSAD). Mitochondrial impairment, including defects in cytochrome oxidase (COX) and diminished mtDNA repair, has been noted in DS cells. The current project encompasses both clinical and cell studies. Firstly, we aim to assess mitochondrial dysfunction in vivo using non-invasive phosphorous magnetic resonance spectroscopy (31P-MRS) in skeletal muscle. Secondly, we aim to investigate the underlying cause of mitochondrial dysfunction in vitro by investigating muscle biopsies. Methodologies and preliminary findings are discussed in this poster. We suggest that neurological consequences of DS including DSAD progression and precocious ageing could be modified by mitochondrial dysfunction. Other characteristics of the syndrome including extremely low levels of activity and impaired exercise capacity could also be affected by mitochondrial inefficiency. Although people with DS have the most to gain from these studies, data will provide information as to the natural history of AD in the general population as DS remains the best and most natural model of AD.

Ion channel drug discovery for demyelinating diseases: a selective inhibitor of Kv1.1-(1.2)3 potassium channels identi ed by high throughput virtual screening and automated patch clamp is e ective in an ex vivo model of axonal demyelination Presenting author: Dr Marc Rogers ([email protected]) Authors: Kirby R, Tang R, Rogers M, Chan F and Madge D http://www.neuroscience.cam.ac.uk/events/abstracts.php?key=e2d28c410e&pw=Submit&event_permalink=50903b958e

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Authors' affiliations: Xention Limited, Iconix Park, Pampisford, Cambridge CB22 3EG, UK Keywords: Neuropharmacology, New Technologies, virtual screening, potassium channel, automated patch clamp, electrophysiology, axonal demyelination Axonal conduction requires potassium (Kv) channels at juxtaparanodal regions and sodium channels at nodes of Ranvier. The main axonal Kv channel is a heteromultimer of one Kv1.1 and three Kv1.2 subunits with unique pharmacology1. The normal distribution of ion channels along peripheral and central axons is disrupted in demyelinating diseases such as multiple sclerosis and Charcot-Marie-Tooth2,3, leading to aberrant action potential firing (pain) and loss of electrical signalling (motor dysfunction, paralysis). Selective antagonists may relieve symptoms with superior efficacy and tolerability than the non-specific inhibitor 4-AP, marketed as Fampridine4. To this end we set out to identify and characterise selective inhibitors of Kv1.1-(1.2)3 channels. The correct axonal Kv1.1-(1.2)3 stoichiometry was stably expressed as a concatamer and tested on the QPatch robotic patch clamp platform. Our virtual screening exercise was described previously5, but briefly 700 compounds from our ion channel focussed library was used with a Kv channel homology model to train computational models and their predictions tested by single point screening. The optimised models then interrogated libraries of drug-like compounds and testing of 250 yielded a 15% hit rate. Pertinent hits were screened by IC50 and Kv1.x gene family selectivity was determined, ranging up to 50 fold. Four hits with a Kv1.1-(1.2)3 IC50