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1.  Clinical, genetic, neurophysiological and functional study of new mutations in episodic ataxia type 1 
Background and objective
Heterozygous mutations in KCNA1 cause episodic ataxia type 1 (EA1), an ion channel disorder characterised by brief paroxysms of cerebellar dysfunction and persistent neuromyotonia. This paper describes four previously unreported families with EA1, with the aim of understanding the phenotypic spectrum associated with different mutations.
15 affected individuals from four families underwent clinical, genetic and neurophysiological evaluation. The functional impact of new mutations identified in the KCNA1 gene was investigated with in vitro electrophysiology and immunocytochemistry.
Detailed clinical documentation, dating back to 1928 in one family, indicates that all patients manifested episodic ataxia of varying severity. Four subjects from three families reported hearing impairment, which has not previously been reported in association with EA1. New mutations (R167M, C185W and I407M) were identified in three out of the four families. When expressed in human embryonic kidney cells, all three new mutations resulted in a loss of Kv1.1 channel function. The fourth family harboured a previously reported A242P mutation, which has not been previously described in association with ataxia.
The genetic basis of EA1 in four families is established and this report presents the earliest documented case from 1928. All three new mutations caused a loss of Kv1.1 channel function. The finding of deafness in four individuals raises the possibility of a link between Kv1.1 dysfunction and hearing impairment. Our findings broaden the phenotypic range associated with mutations in KCNA1.
PMCID: PMC4332158  PMID: 23349320
Cerebellar Ataxia; Epilepsy; Neurogenetics; Neuromuscular; Neurophysiol, Clinical
2.  What's wrong with the amygdala in temporal lobe epilepsy? 
Brain  2011;134(10):2800-2801.
PMCID: PMC3187545  PMID: 21921018
3.  Ionotropic receptors at hippocampal mossy fibers: roles in axonal excitability, synaptic transmission, and plasticity 
Dentate granule cells process information from the enthorinal cortex en route to the hippocampus proper. These neurons have a very negative resting membrane potential and are relatively silent in the slice preparation. They are also subject to strong feed-forward inhibition. Their unmyelinated axon or mossy fiber ramifies extensively in the hilus and projects to stratum lucidum where it makes giant en-passant boutons with CA3 pyramidal neurons. There is compelling evidence that mossy fiber boutons express presynaptic GABAA receptors, which are commonly found in granule cell dendrites. There is also suggestive evidence for the presence of other ionotropic receptors, including glycine, NMDA, and kainate receptors, in mossy fiber boutons. These presynaptic receptors have been proposed to lead to mossy fiber membrane depolarization. How this phenomenon alters the excitability of synaptic boutons, the shape of presynaptic action potentials, Ca2+ influx and neurotransmitter release has remained elusive, but high-resolution live imaging of individual varicosities and direct patch-clamp recordings have begun to shed light on these phenomena. Presynaptic GABAA and kainate receptors have also been reported to facilitate the induction of long-term potentiation at mossy fiber—CA3 synapses. Although mossy fibers are highly specialized, some of the principles emerging at this connection may apply elsewhere in the CNS.
PMCID: PMC3540408  PMID: 23316138
2-photon microscopy; GABAA receptor; granule cell; immunogold; kainate receptor; mossy fiber bouton; NMDA receptor; single channel
4.  Differential triggering of spontaneous glutamate release by P/Q-, N-, and R-type Ca2+ channels 
Nature neuroscience  2013;16(12):1754-1763.
The role of voltage-gated Ca2+ channels (VGCCs) in spontaneous miniature neurotransmitter release is incompletely understood. Here we show that stochastic opening of P/Q-, N-, and R-type VGCCs accounts for ~50% of all spontaneous glutamate release at rat cultured hippocampal synapses, and that R-type channels play a far greater role in spontaneous than in action potential-evoked exocytosis. VGCC-dependent ‘minis’ show similar sensitivity to presynaptic Ca2+ chelation as evoked release, arguing for direct triggering of spontaneous release by transient spatially localized Ca2+ domains. Experimentally constrained three-dimensional diffusion modeling of Ca2+ influx-exocytosis coupling is consistent with clustered distribution of VGCCs in the active zone of small hippocampal synapses, and shows that spontaneous VGCCs openings can account for the experimentally observed VGCC-dependent minis, although single channel openings trigger release with low probability. Uncorrelated stochastic VGCC opening is thus a major trigger for spontaneous glutamate release, with differential roles for distinct channel subtypes.
PMCID: PMC4176737  PMID: 24185424
5.  Cholinergic Axons Modulate GABAergic Signaling among Hippocampal Interneurons via Postsynaptic α7 Nicotinic Receptors 
Homopentameric α7 nicotinic receptors have a high affinity for acetylcholine (ACh), are permeable to Ca2+ ions, and are abundant in hippocampal interneurons. Although nicotinic agonists evoke inward currents and Ca2+ transients in stratum radiatum interneurons, the role of endogenous ACh in modulating synaptic integration by interneurons is incompletely understood. Many cholinergic axonal varicosities do not have postsynaptic specializations, but α7 receptors frequently occur close to synaptic GABAA receptors. These observations raise the possibility that α7 nicotinic receptors activated by ACh released from cholinergic axons modulate GABAergic transmission in interneurons. We show that agonists of α7 receptors profoundly depress GABAergic IPSCs recorded in stratum radiatum interneurons in the CA1 region of the hippocampus. This depression is accompanied by a small increase in GABA release. α7 nicotinic receptor agonists also depress GABA- or muscimol-evoked currents in interneurons, indicating that the major effect is a postsynaptic modulation of GABAA receptors. The depression of GABA-evoked currents is abolished by chelating Ca2+ in the recorded interneuron and attenuated by inhibitors of PKC. We also show that stimuli designed to release endogenous ACh from cholinergic axons evoke an α7 receptor-dependent heterosynaptic depression of GABAergic IPSCs in interneurons. This heterosynaptic modulation is amplified by blocking cholinesterases. These results reveal a novel mechanism by which cholinergic neurons modulate information processing in the hippocampus.
PMCID: PMC2889598  PMID: 17522313
nicotinic; GABAA receptor; interneurons; cholinergic; hippocampus; inhibition
6.  Long-term potentiation in hippocampal oriens interneurons: postsynaptic induction, presynaptic expression and evaluation of candidate retrograde factors 
Several types of hippocampal interneurons exhibit a form of long-term potentiation (LTP) that depends on Ca2+-permeable AMPA receptors and group I metabotropic glutamate receptors. Several sources of evidence point to a presynaptic locus of LTP maintenance. The retrograde factor that triggers the expression of LTP remains unidentified. Here, we show that trains of action potentials in putative oriens-lacunosum-moleculare interneurons of the mouse CA1 region can induce long-lasting potentiation of stimulus-evoked excitatory postsynaptic currents that mimics LTP elicited by high-frequency afferent stimulation. We further report that blockers of nitric oxide production or TRPV1 receptors failed to prevent LTP induction. The present results add to the evidence that retrograde signalling underlies N-methyl-d-aspartate (NMDA) receptor-independent LTP in oriens interneurons, mediated by an unidentified factor.
PMCID: PMC3843866  PMID: 24298136
long-term potentiation; interneurons; anti-Hebbian
7.  In vivo loss of slow potassium channel activity in individuals with benign familial neonatal epilepsy in remission 
Brain  2012;135(10):3144-3152.
Benign familial neonatal epilepsy is a neuronal channelopathy most commonly caused by mutations in KCNQ2, which encodes the Kv7.2 subunit of the slow K+ channel. Kv7.2 is expressed in both central and peripheral nervous systems. Seizures occur in the neonatal period, often in clusters within the first few days of life, and usually remit by 12 months of age. The mechanism of involvement of Kv7.2 mutations in the process of seizure generation has not been established in vivo. In peripheral axons, Kv7.2 contributes to the nodal slow K+ current. The present study aimed to determine whether axonal excitability studies could detect changes in peripheral nerve function related to dysfunction or loss of slow potassium channel activity. Nerve excitability studies were performed on eight adults with KCNQ2 mutations and a history of benign familial neonatal epilepsy, now in remission. Studies detected distinctive changes in peripheral nerve, indicating a reduction in slow K+ current. Specifically, accommodation to long-lasting depolarizing currents was reduced in mutation carriers by 24% compared with normal controls, and the threshold undershoot after 100 ms depolarizing currents was reduced by 22%. Additional changes in excitability included a reduction in the relative refractory period, an increase in superexcitability and a tendency towards reduced sub-excitability. Modelling of the nerve excitability changes suggested that peripheral nerve hyperexcitability may have been ameliorated by upregulation of other potassium channels. We conclude that subclinical dysfunction of Kv7.2 in peripheral axons can be reliably detected non-invasively in adulthood. Related alterations in neuronal excitability may contribute to epilepsy associated with KCNQ2 mutations.
PMCID: PMC3470715  PMID: 23065794
epilepsy; channelopathy; nerve excitability; neuromyotonia; potassium channel
8.  Nanoscale-Targeted Patch-Clamp Recordings of Functional Presynaptic Ion Channels 
Neuron  2013;79(6):1067-1077.
Direct electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤1 μm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100–150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell). Using this technique, we report presynaptic recordings of K+, Na+, Cl−, and Ca2+ channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses.
Video Abstract
•Topographic imaging of live synaptic boutons at nanoscale resolution•Cell-attached patch-clamp recordings of ion channels in small central synapses•Whole-cell small presynaptic bouton recordings
Novak and colleagues have developed a method for nanoscale-targeted patch-clamp presynaptic recordings in submicrometer central synapses identified using superresolution scanning ion conductance microscopy. This semiautomated approach opens a new window on the physiology of small presynaptic terminals.
PMCID: PMC3781326  PMID: 24050398
9.  Optogenetic and Potassium Channel Gene Therapy in a Rodent Model of Focal Neocortical Epilepsy 
Science translational medicine  2012;4(161):161ra152.
Neocortical epilepsy is frequently drug-resistant. Surgery to remove the epileptogenic zone is only feasible in a minority of cases, leaving many patients without an effective treatment. We report the potential efficacy of gene therapy in focal neocortical epilepsy using a rodent model in which epilepsy is induced by tetanus toxin injection in the motor cortex. By applying several complementary methods that use continuous wireless electroencephalographic monitoring to quantify epileptic activity, we observed increases in high frequency activity and in the occurrence of epileptiform events. Pyramidal neurons in the epileptic focus showed enhanced intrinsic excitability consistent with seizure generation. Optogenetic inhibition of a subset of principal neurons transduced with halorhodopsin targeted to the epileptic focus by lentiviral delivery was sufficient to attenuate electroencephalographic seizures. Local lentiviral overexpression of the potassium channel Kv1.1 reduced the intrinsic excitability of transduced pyramidal neurons. Coinjection of this Kv1.1 lentivirus with tetanus toxin fully prevented the occurrence of electroencephalographic seizures. Finally, administration of the Kv1.1 lentivirus to an established epileptic focus progressively suppressed epileptic activity over several weeks without detectable behavioral side effects. Thus, gene therapy in a rodent model can be used to suppress seizures acutely, prevent their occurrence after an epileptogenic stimulus, and successfully treat established focal epilepsy.
PMCID: PMC3605784  PMID: 23147003
10.  Complementary roles of intra- and extra-synaptic NMDA receptors in homeostatic synaptic plasticity? 
Science (New York, N.Y.)  2004;305(5692):1912-reply.
Liu et al (Science, 304:1021-4) report that NMDA receptors containing NR2A and NR2B subunits are selectively coupled to long-term potentiation (LTP) and long-term depression (LTD) respectively. Because NR2B (but not NR2A) receptors occur outside synapses, and can be activated by glutamate spillover, this principle may underlie synaptic homeostasis.
PMCID: PMC1410731  PMID: 15448254
11.  Efficient “Communication through Coherence” Requires Oscillations Structured to Minimize Interference between Signals 
PLoS Computational Biology  2012;8(11):e1002760.
The ‘communication through coherence’ (CTC) hypothesis proposes that selective communication among neural networks is achieved by coherence between firing rate oscillation in a sending region and gain modulation in a receiving region. Although this hypothesis has stimulated extensive work, it remains unclear whether the mechanism can in principle allow reliable and selective information transfer. Here we use a simple mathematical model to investigate how accurately coherent gain modulation can filter a population-coded target signal from task-irrelevant distracting inputs. We show that selective communication can indeed be achieved, although the structure of oscillatory activity in the target and distracting networks must satisfy certain previously unrecognized constraints. Firstly, the target input must be differentiated from distractors by the amplitude, phase or frequency of its oscillatory modulation. When distracting inputs oscillate incoherently in the same frequency band as the target, communication accuracy is severely degraded because of varying overlap between the firing rate oscillations of distracting inputs and the gain modulation in the receiving region. Secondly, the oscillatory modulation of the target input must be strong in order to achieve a high signal-to-noise ratio relative to stochastic spiking of individual neurons. Thus, whilst providing a quantitative demonstration of the power of coherent oscillatory gain modulation to flexibly control information flow, our results identify constraints imposed by the need to avoid interference between signals, and reveal a likely organizing principle for the structure of neural oscillations in the brain.
Author Summary
Distributed regions of mammalian brains transiently engage in coherent oscillations, often at specific stages of behavioral or cognitive tasks. This activity may play a role in controlling information flow among connected regions, allowing the brain's connectivity structure to be flexibly reconfigured in response to changing task demands. We have used a computational model to investigate the conditions under which oscillations can generate selective communication through a mechanism in which the excitability of neurons in one region is modulated coherently with a firing rate oscillation in another region. Our results demonstrate that this mechanism is able to accurately and selectively control the flow of signals encoded as spatial patterns of firing rate. However, we found that the requirement to avoid interference between different signals imposes previously unrecognised constraints on the structures of oscillatory activity that can efficiently support this mechanism. These constraints may be an organizing principle for the structured oscillatory activity observed in vivo.
PMCID: PMC3493486  PMID: 23144603
12.  Oscillatory dynamics in the hippocampus support dentate gyrus–CA3 coupling 
Nature neuroscience  2012;15(5):763-768.
Gamma oscillations in the dentate gyrus and hippocampal CA3 show variable coherence in vivo, but the mechanisms and relevance for information flow are unknown. We found that carbachol-induced oscillations in rat CA3 have biphasic phase-response curves, consistent with the ability to couple with oscillations in afferent projections. Differences in response to stimulation of either the intrinsic feedback circuit or the dentate gyrus were well described by varying an impulse vector in a two-dimensional dynamical system, representing the relative input to excitatory and inhibitory neurons. Responses to sinusoidally modulated optogenetic stimulation confirmed that the CA3 network oscillation can entrain to periodic inputs, with a steep dependence of entrainment phase on input frequency. CA3 oscillations are therefore suited to coupling with oscillations in the dentate gyrus over a broad range of frequencies.
PMCID: PMC3378654  PMID: 22466505
13.  Independent Regulation of Basal Neurotransmitter Release Efficacy by Variable Ca2+ Influx and Bouton Size at Small Central Synapses 
PLoS Biology  2012;10(9):e1001396.
Concurrent imaging of vesicular release and calcium dynamics in small presynaptic boutons shows that the fusion probability of readily releasable vesicles is a major determinant of the overall variability in release probability.
The efficacy of action potential evoked neurotransmitter release varies widely even among synapses supplied by the same axon, and the number of release-ready vesicles at each synapse is a major determinant of this heterogeneity. Here we identify a second, equally important, mechanism for release heterogeneity at small hippocampal synapses, the inter-synaptic variation of the exocytosis probability of release-ready vesicles. Using concurrent measurements of vesicular pool sizes, vesicular exocytosis rates, and presynaptic Ca2+ dynamics, in the same small hippocampal boutons, we show that the average fusion probability of release-ready vesicles varies among synapses supplied by the same axon with the size of the spike-evoked Ca2+ concentration transient. We further show that synapses with a high vesicular release probability exhibit a lower Ca2+ cooperativity, arguing that this is a direct consequence of increased Ca2+ influx at the active zone. We conclude that variability of neurotransmitter release under basal conditions at small central synapses is accounted for not only by the number of release-ready vesicles, but also by their fusion probabilities, which are set independently of bouton size by variable spike-evoked presynaptic Ca2+ influx.
PMCID: PMC3457933  PMID: 23049481
14.  Presynaptic fluctuations and release-independent depression 
Nature neuroscience  2006;9(9):1091-1093.
Although vesicle depletion contributes to short-term depression, several studies have reported that the release probability can be transiently depressed even if an action potential fails to evoke release. Here we argue that stochastic fluctuation in the release machinery can give rise to apparent release-independent depression as a result of sampling bias. The relationship between this apparent depression and the interstimulus interval provides a window on the kinetics of state transitions of the release apparatus.
PMCID: PMC3433797  PMID: 16878129
15.  NR2B-Containing Receptors Mediate Cross Talk among Hippocampal Synapses 
Under some conditions, synaptically released glutamate can exert long-range actions in the cortical microcircuitry. To what extent glutamate spillover leads to direct cross talk among individual synapses remains unclear. We recorded NMDAR-mediated EPSCs in acute hippocampal slices at 35°C by stimulating two independent pathways that converge on the same CA1 pyramidal cell. Activation of a conditioning pathway in the presence of the use-dependent blocker dizocilpine maleate (MK801) resulted in partial NMDA receptor (NMDAR) blockade in the other, silent pathway. This was accompanied by an increase in the rise time of the EPSCs in the conditioning (although not the silent) pathway, implying an increase in diffusional distance from release site to NMDARs. We estimated that up to ~30% of NMDARs contributing to EPSCs were activated by glutamate released from multiple synaptic sources; however, NMDAR-mediated synaptic cross talk was undetectable when NR2B subunit-containing receptors were blocked (but could be rescued by blocking glutamate uptake). We propose that NR2B-containing NMDARs can detect glutamate arising from multiple synapses, whereas NR2A-containing NMDARs only normally mediate direct synaptic transmission. These NMDAR isoforms thus play complementary roles in sensing global and local glutamate signals, respectively.
PMCID: PMC3379686  PMID: 15152037
hippocampus; glutamate; spillover; NMDA receptors; NR2B; synapse
16.  Anti-Hebbian Long-Term Potentiation in the Hippocampal Feedback Inhibitory Circuit 
Science (New York, N.Y.)  2007;315(5816):1262-1266.
Long-term potentiation (LTP), which approximates Hebb’s postulate of associative learning, typically requires depolarization-dependent glutamate receptors of the NMDA (N-methyl-d-aspartate) subtype. However, in some neurons, LTP depends instead on calcium-permeable AMPA-type receptors. This is paradoxical because intracellular polyamines block such receptors during depolarization. We report that LTP at synapses on hippocampal interneurons mediating feedback inhibition is “anti-Hebbian”: It is induced by presynaptic activity but prevented by postsynaptic depolarization. Anti-Hebbian LTP may occur in interneurons that are silent during periods of intense pyramidal cell firing, such as sharp waves, and lead to their altered activation during theta activity.
PMCID: PMC3369266  PMID: 17332410
17.  Presynaptic, extrasynaptic and axonal GABAA receptors in the CNS: where and why? 
Although GABAA receptors are widely distributed at inhibitory synapses on dendrites and cell bodies of neurons, they also occur in other places, in particular at synapses made on axons and in extrasynaptic membranes. This review summarises some of the evidence that presynaptic receptors modulate transmission not only at primary afferents in the spinal cord, but also at a variety of sites in the brain, including hippocampal mossy fibres. These receptors modulate transmitter release via several different mechanisms. Another form of unconventional GABAA receptor-mediated signalling is the mediation of a tonic conductance, seen in granule cells of the cerebellum and dentate gyrus and also in hippocampal interneurons. Tonic signalling appears to be mediated by extrasynaptic receptors. The adaptive significance of this form of signalling remains poorly understood.
PMCID: PMC3369532  PMID: 15471589
Presynaptic inhibition; GABA receptors; Tonic; Phasic; Subunit
18.  GABAA Receptors at Hippocampal Mossy Fibers 
Neuron  2003;39(6):961-973.
Presynaptic GABAA receptors modulate synaptic transmission in several areas of the CNS but are not known to have this action in the cerebral cortex. We report that GABAA receptor activation reduces hippocampal mossy fibers excitability but has the opposite effect when intracellular Cl− is experimentally elevated. Synaptically released GABA mimics the effect of exogenous agonists. GABAA receptors modulating axonal excitability are tonically active in the absence of evoked GABA release or exogenous agonist application. Presynaptic action potential-dependent Ca2+ transients in individual mossy fiber varicosities exhibit a biphasic dependence on membrane potential and are altered by GABAA receptors. Antibodies against the α2 subunit of GABAA receptors stain mossy fibers. Axonal GABAA receptors thus play a potentially important role in tonic and activity-dependent heterosynaptic modulation of information flow to the hippocampus.
PMCID: PMC3368315  PMID: 12971896
19.  Nerve excitability studies characterize KV1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1 
Brain  2010;133(12):3530-3540.
Episodic ataxia type 1 is a neuronal channelopathy caused by mutations in the KCNA1 gene encoding the fast K+ channel subunit Kv1.1. Episodic ataxia type 1 presents with brief episodes of cerebellar dysfunction and persistent neuromyotonia and is associated with an increased incidence of epilepsy. In myelinated peripheral nerve, Kv1.1 is highly expressed in the juxtaparanodal axon, where potassium channels limit the depolarizing afterpotential and the effects of depolarizing currents. Axonal excitability studies were performed on patients with genetically confirmed episodic ataxia type 1 to characterize the effects of Kv1.1 dysfunction on motor axons in vivo. The median nerve was stimulated at the wrist and compound muscle action potentials were recorded from abductor pollicis brevis. Threshold tracking techniques were used to record strength-duration time constant, threshold electrotonus, current/threshold relationship and the recovery cycle. Recordings from 20 patients from eight kindreds with different KCNA1 point mutations were compared with those from 30 normal controls. All 20 patients had a history of episodic ataxia and 19 had neuromyotonia. All patients had similar, distinctive abnormalities: superexcitability was on average 100% higher in the patients than in controls (P < 0.00001) and, in threshold electrotonus, the increase in excitability due to a depolarizing current (20% of threshold) was 31% higher (P < 0.00001). Using these two parameters, the patients with episodic ataxia type 1 and controls could be clearly separated into two non-overlapping groups. Differences between the different KCNA1 mutations were not statistically significant. Studies of nerve excitability can identify Kv1.1 dysfunction in patients with episodic ataxia type 1. The simple 15 min test may be useful in diagnosis, since it can differentiate patients with episodic ataxia type 1 from normal controls with high sensitivity and specificity.
PMCID: PMC2995887  PMID: 21106501
ataxia; channelopathy; nerve excitability; neuromyotonia; potassium channel
20.  mGluR agonist-evoked LTP in hippocampal oriens interneurons 
Several subtypes of interneurons in the feedback circuit in stratum oriens of the hippocampus exhibit NMDA receptor-independent long-term potentiation (LTP) at glutamatergic synapses made by local pyramidal neurons. LTP has been reported with both ‘Hebbian’ and ‘anti-Hebbian’ induction protocols, where high-frequency presynaptic stimulation is paired with either postsynaptic depolarization or hyperpolarization. Do these phenomena represent distinct forms of plasticity, dependent on group I metabotropic receptors (mGluRs) and rectifying Ca2+-permeable AMPA receptors, respectively? Blockade of either mGluR1 or mGluR5 prevented anti-Hebbian LTP induction in stratum oriens interneurons in rat hippocampal slices. Exogenous activation of group I mGluRs by the selective agonist (s)-3,5-dihydroxyphenylglycine (DHPG) was unable to induce LTP on its own, and instead depressed excitatory transmission. However, when paired with postsynaptic hyperpolarization, DHPG, or the mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), elicited a delayed long-lasting potentiation, which was accompanied by a decrease in paired-pulse facilitation. Anti-Hebbian LTP occluded the effect of DHPG paired with hyperpolarization, implying that the induction cascades triggered by both conjunctions of stimuli converge on common expression mechanisms.
PMCID: PMC3083832  PMID: 21490219
hippocampus; interneuron; metabotropic glutamate receptors; LTP
21.  Alternative Splicing Modulates Inactivation of Type 1 Voltage-gated Sodium Channels by Toggling an Amino Acid in the First S3-S4 Linker* 
The Journal of Biological Chemistry  2011;286(42):36700-36708.
Background: Small changes in voltage-gated sodium channel behavior can disrupt neuronal activity and cause severe neurological disorders.
Results: By changing a single amino acid, a conserved alternative splicing event modifies the stability of channel inactivation.
Conclusion: Splicing can regulate inactivation of sodium channels.
Significance: Many commonly used drugs target sodium channel inactivation; consequently, splicing could affect treatment of several neurological disorders.
Voltage-gated sodium channels underlie the upstroke of action potentials and are fundamental to neuronal excitability. Small changes in the behavior of these channels are sufficient to change neuronal firing and trigger seizures. These channels are subject to highly conserved alternative splicing, affecting the short linker between the third transmembrane segment (S3) and the voltage sensor (S4) in their first domain. The biophysical consequences of this alternative splicing are incompletely understood. Here we focus on type 1 sodium channels (Nav1.1) that are implicated in human epilepsy. We show that the functional consequences of alternative splicing are highly sensitive to recording conditions, including the identity of the major intracellular anion and the recording temperature. In particular, the inactivation kinetics of channels containing the alternate exon 5N are more sensitive to intracellular fluoride ions and to changing temperature than channels containing exon 5A. Moreover, Nav1.1 channels containing exon 5N recover from inactivation more rapidly at physiological temperatures. Three amino acids differ between exons 5A and 5N. However, the changes in sensitivity and stability of inactivation were reproduced by a single conserved change from aspartate to asparagine in channels containing exon 5A, which was sufficient to make them behave like channels containing the complete exon 5N sequence. These data suggest that splicing at this site can modify the inactivation of sodium channels and reveal a possible interaction between splicing and anti-epileptic drugs that stabilize sodium channel inactivation.
PMCID: PMC3196094  PMID: 21890636
Ion Channels; Membrane Proteins; Neurobiology; Neurological Diseases; RNA Splicing; Sodium Channels; HEK Cells; Patch Clamp; Voltage-dependent
22.  Presynaptic GABAA receptors enhance transmission and LTP induction at hippocampal mossy fiber synapses 
Nature neuroscience  2010;13(4):431-438.
Presynaptic GABAA receptors occur at hippocampal mossy fiber synapses. Whether and how they modulate orthodromic signaling to postsynaptic targets is poorly understood. We show that an endogenous neurosteroid selective for high-affinity δ-subunit-containing GABAA receptors depolarizes rat mossy fiber boutons, enhances action potential-dependent Ca2+ transients, and facilitates glutamatergic transmission to pyramidal neurons. Conversely, blocking GABAA receptors hyperpolarizes mossy fiber boutons, increases their input resistance, decreases spike width, and attenuates action potential-dependent presynaptic Ca2+ transients, indicating that a subset of presynaptic GABA receptors are tonically active. Blocking GABAA receptors also interferes with the induction of long-term potentiation at mossy fiber-CA3 synapses. Presynaptic GABAA receptors thus facilitate information flow to the hippocampus both directly and by enhancing LTP.
PMCID: PMC2898498  PMID: 20305647
23.  Oscillations and Filtering Networks Support Flexible Routing of Information 
Neuron  2010;67(2-10):308-320.
The mammalian brain exhibits profuse interregional connectivity. How information flow is rapidly and flexibly switched among connected areas remains poorly understood. Task-dependent changes in the power and interregion coherence of network oscillations suggest that such oscillations play a role in signal routing. We show that switching one of several convergent pathways from an asynchronous to an oscillatory state allows accurate selective transmission of population-coded information, which can be extracted even when other convergent pathways fire asynchronously at comparable rates. We further show that the band-pass filtering required to perform this information extraction can be implemented in a simple spiking network model with a single feed-forward interneuron layer. This constitutes a mechanism for flexible signal routing in neural circuits, which exploits sparsely synchronized network oscillations and temporal filtering by feed-forward inhibition.
Video Abstract
► We report a mechanism for selective routing of population-coded signals ► Spatial rate codes can be reproduced as oscillation amplitude patterns ► A feed-forward interneuron layer can be tuned to act as a band-pass filter ► We show how multiple population codes can be represented in parallel by oscillations
PMCID: PMC3125699  PMID: 20670837
24.  N-methyl-d-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes 
Brain  2010;133(6):1655-1667.
Antibodies to the N-methyl-d-aspartate subtype of glutamate receptor have been associated with a newly-described encephalopathy that has been mainly identified in young females with ovarian tumours. However, the full clinical spectrum and treatment responses are not yet clear. We established a sensitive cell-based assay for detection of N-methyl-d-aspartate receptor antibodies in serum or cerebrospinal fluid, and a quantitative fluorescent immunoprecipitation assay for serial studies. Although there was marked intrathecal synthesis of N-methyl-d-aspartate receptor antibodies, the absolute levels of N-methyl-d-aspartate receptor antibodies were higher in serum than in cerebrospinal fluid. N-methyl-d-aspartate receptor antibodies were of the immunoglobulin G1 subclass and were able to activate complement on N-methyl d-aspartate receptor-expressing human embryonic kidney cells. From questionnaires returned on 44 N-methyl-d-aspartate receptor antibody-positive patients, we identified a high proportion without a detected tumour (35/44, 80%: follow-up 3.6–121 months, median 16 months). Among the latter were 15 adult females (43%), 10 adult males (29%) and 10 children (29%), with four in the first decade of life. Overall, there was a high proportion (29%) of non-Caucasians. Good clinical outcomes, as defined by reductions in modified Rankin scores, correlated with decreased N-methyl-d-aspartate receptor antibody levels and were associated with early (<40 days) administration of immunotherapies in non-paraneoplastic patients (P < 0.0001) and earlier tumour removal in paraneoplastic patients (P = 0.02). Ten patients (23%) who were first diagnosed during relapses had no evidence of tumours but had received minimal or no immunotherapy during earlier episodes. Temporal analysis of the onset of the neurological features suggested progression through two main stages. The time of onset of the early features, characterized by neuropsychiatric symptoms and seizures preceded by a median of 10–20 days, the onset of movement disorders, reduction in consciousness and dysautonomia. This temporal dichotomy was also seen in the timing of cerebrospinal fluid, electroencephalographic and in the rather infrequent cerebral imaging changes. Overall, our data support a model in which the early features are associated with cerebrospinal fluid lymphocytosis, and the later features with appearance of oligoclonal bands. The immunological events and neuronal mechanisms underlying these observations need to be explored further, but one possibility is that the early stage represents diffusion of serum antibodies into the cortical grey matter, whereas the later stage results from secondary expansion of the immunological repertoire within the intrathecal compartment acting on subcortical neurons. Four patients, who only had temporal lobe epilepsy without oligoclonal bands, may represent restriction to the first stage.
PMCID: PMC2877907  PMID: 20511282
N-methyl-d-aspartate receptor-antibody encephalitis; autoimmunity; non-paraneoplastic; paraneoplastic; immunotherapy-responsive
25.  Dysfunction of the CaV2.1 calcium channel in cerebellar ataxias 
Mutations in the CACNA1A gene are associated with episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6). CACNA1A encodes the α-subunit of the P/Q-type calcium channel or CaV2.1, which is highly enriched in the cerebellum. It is one of the main channels linked to synaptic transmission throughout the human central nervous system. Here, we compare recent advances in the understanding of the genetic changes that underlie EA2 and SCA6 and what these new findings suggest about the mechanism of the disease.
PMCID: PMC2948357  PMID: 20948794

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