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.
Cerebellar Ataxia; Epilepsy; Neurogenetics; Neuromuscular; Neurophysiol, Clinical
Brain development and interictal function are unaffected in many paroxysmal neurological channelopathies, possibly explained by homoeostatic plasticity of synaptic transmission. Episodic ataxia type 1 is caused by missense mutations of the potassium channel Kv1.1, which is abundantly expressed in the terminals of cerebellar basket cells. Presynaptic action potentials of small inhibitory terminals have not been characterized, and it is not known whether developmental plasticity compensates for the effects of Kv1.1 dysfunction. Here we use visually targeted patch-clamp recordings from basket cell terminals of mice harbouring an ataxia-associated mutation and their wild-type littermates. Presynaptic spikes are followed by a pronounced afterdepolarization, and are broadened by pharmacological blockade of Kv1.1 or by a dominant ataxia-associated mutation. Somatic recordings fail to detect such changes. Spike broadening leads to increased Ca2+ influx and GABA release, and decreased spontaneous Purkinje cell firing. We find no evidence for developmental compensation for inherited Kv1.1 dysfunction.
Episodic ataxia type 1 is caused by mutations in the potassium channel Kv1.1, which is found in cerebellar basket cells. Here, the authors use electrophysiology techniques to characterize these mutant channels, and observe that the changes result in decreased spontaneous Purkinje cell firing with no evidence for developmental compensation.
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.
2-photon microscopy; GABAA receptor; granule cell; immunogold; kainate receptor; mossy fiber bouton; NMDA receptor; single channel
Episodic ataxia type 2 is caused by mutations in CACNA1A, which encodes the
pore-forming subunit of the voltage-gated calcium channel Cav2.1.
Tomlinson et al. reveal altered peripheral axonal
excitability in patients compared to controls. The changes may reflect malformation
of the nodes of Ranvier, as demonstrated in animal models.
Episodic ataxia type 2 is caused by mutations in CACNA1A, which encodes the
pore-forming subunit of the voltage-gated calcium channel Cav2.1.
Tomlinson et al. reveal altered peripheral
axonal excitability in patients compared to controls. The changes may
reflect malformation of the nodes of Ranvier, as demonstrated in animal
Ion channel dysfunction causes a range of neurological disorders by altering
transmembrane ion fluxes, neuronal or muscle excitability, and neurotransmitter
release. Genetic neuronal channelopathies affecting peripheral axons provide a unique
opportunity to examine the impact of dysfunction of a single channel subtype in
detail in vivo. Episodic ataxia type 2 is caused by
mutations in CACNA1A, which encodes the pore-forming
subunit of the neuronal voltage-gated calcium channel Cav2.1. In
peripheral motor axons, this channel is highly expressed at the presynaptic
neuromuscular junction where it contributes to action potential-evoked
neurotransmitter release, but it is not expressed mid-axon or thought to contribute
to action potential generation. Eight patients from five families with genetically
confirmed episodic ataxia type 2 underwent neurophysiological assessment to determine
whether axonal excitability was normal and, if not, whether changes could be
explained by Cav2.1 dysfunction. New mutations in the CACNA1A gene were identified in two families. Nerve conduction studies
were normal, but increased jitter in single-fibre EMG studies indicated unstable
neuromuscular transmission in two patients. Excitability properties of median motor
axons were compared with those in 30 age-matched healthy control subjects. All
patients had similar excitability abnormalities, including a high electrical
threshold and increased responses to hyperpolarizing (P
< 0.00007) and depolarizing currents (P < 0.001)
in threshold electrotonus. In the recovery cycle, refractoriness (P < 0.0002) and superexcitability (P < 0.006) were increased. Cav2.1 dysfunction in episodic
ataxia type 2 thus has unexpected effects on axon excitability, which may reflect an
indirect effect of abnormal calcium current fluxes during development.
Episodic ataxia type 2 is caused by mutations in CACNA1A, which encodes the pore-forming subunit of the voltage-gated
calcium channel Cav2.1. Tomlinson et al.
reveal altered peripheral axonal excitability in patients compared to controls. The
changes may reflect malformation of the nodes of Ranvier, as demonstrated in animal
channelopathy; axonal excitability
Mammalian brains exhibit population oscillations whose structures vary in time and space according to behavioural state. A proposed function of these oscillations is to control the flow of signals among anatomically connected networks. However, the nature of neural coding that may support oscillatory selective communication has received relatively little attention. Here we consider the role of multiplexing, whereby multiple information streams share a common neural substrate. We suggest that multiplexing implemented through periodic modulation of firing rate population codes enables flexible reconfiguration of effective connectivity among brain areas.
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.
nicotinic; GABAA receptor; interneurons; cholinergic; hippocampus; inhibition
An anti-Hebbian form of LTP is observed at excitatory synapses made with some hippocampal interneurons. LTP induction is facilitated when postsynaptic interneurons are hyperpolarized, presumably because Ca2+ entry through Ca2+-permeable glutamate receptors is enhanced. The contribution of modulatory transmitters to anti-Hebbian LTP induction remains to be established. Activation of group I metabotropic receptors (mGluRs) is required for anti-Hebbian LTP induction in interneurons with cell bodies in the CA1 stratum oriens. This region receives a strong cholinergic innervation from the septum, and muscarinic acetylcholine receptors (mAChRs) share some signaling pathways and cooperate with mGluRs in the control of neuronal excitability.
We therefore examined possible interactions between group I mGluRs and mAChRs in anti-Hebbian LTP at synapses which excite oriens interneurons in rat brain slices. We found that blockade of either group I mGluRs or M1 mAChRs prevented the induction of anti-Hebbian LTP by pairing presynaptic activity with postsynaptic hyperpolarization. Blocking either receptor also suppressed long-term effects of activation of the other G-protein coupled receptor on interneuron membrane potential. However, no crossed blockade was detected for mGluR or mAchR effects on interneuron after-burst potentials or on the frequency of miniature EPSPs. Paired recordings between pyramidal neurons and oriens interneurons were obtained to determine whether LTP could be induced without concurrent stimulation of cholinergic axons. Exogenous activation of mAChRs led to LTP, with changes in EPSP amplitude distributions consistent with a presynaptic locus of expression. LTP, however, required noninvasive presynaptic and postsynaptic recordings.
SIGNIFICANCE STATEMENT In the hippocampus, a form of NMDA receptor-independent long-term potentiation (LTP) occurs at excitatory synapses made on some inhibitory neurons. This is preferentially induced when postsynaptic interneurons are hyperpolarized, depends on Ca2+ entry through Ca2+-permeable AMPA receptors, and has been labeled anti-Hebbian LTP. Here we show that this form of LTP also depends on activation of both group I mGluR and M1 mAChRs. We demonstrate that these G-protein coupled receptors (GPCRs) interact, because the blockade of one receptor suppresses long-term effects of activation of the other GPCR on both LTP and interneuron membrane potential. This LTP was also detected in paired recordings, although only when both presynaptic and postsynaptic recordings did not perturb the intracellular medium. Changes in EPSP amplitude distributions in dual recordings were consistent with a presynaptic locus of expression.
hippocampus; interneuron; LTP; metabotropic glutamate receptors; muscarinic receptors
The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.
The potassium-chloride co-transporter, KCC2 is an essential component in maintaining a gradient for chloride ions in neurons. Here Stodberg and colleagues identify loss-of-function mutations in the encoding gene SLC12A5, which impair normal synaptic function associated with early-onset epilepsy.
To determine whether immunoglobulin G (IgG) from patients with Lambert-Eaton myasthenic syndrome (LEMS) decreases action potential–evoked synaptic vesicle exocytosis, and whether the effect is mediated by P/Q-type voltage-gated calcium channels (VGCCs).
IgG was obtained from 4 patients with LEMS (3 males, 1 female), including 2 patients with lung malignancy. Antibodies against P/Q-type VGCCs were detected in all 4 patients, and against N-type VGCCs in 2. We incubated neuronal cultures with LEMS IgG and determined the size of the total recycling pool of synaptic vesicles and the rate of action potential–evoked exocytosis using fluorescence imaging of the amphiphilic dye SynaptoRed C1. Pooled IgG from healthy volunteers was used as a control. We repeated the experiments on synapses lacking P/Q-type calcium channels from a Cacna1a knockout mouse to determine whether these channels account for the pathogenic effect of LEMS IgG.
LEMS IgG had no effect on the total recycling pool size but significantly reduced the rate of action potential–evoked synaptic exocytosis in wild-type neurons when compared with neurons treated with control IgG. In contrast, LEMS IgG had no effect on the rate of synaptic vesicle exocytosis in neurons lacking P/Q-type channels.
These data provide direct evidence that LEMS IgG inhibits neurotransmitter release by acting on P/Q-type VGCCs.
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.
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.
long-term potentiation; interneurons; anti-Hebbian
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.
epilepsy; channelopathy; nerve excitability; neuromyotonia; potassium channel
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.
•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.
The proline-rich transmembrane protein (PRRT2) gene was recently identified using exome sequencing as the cause of autosomal dominant paroxysmal kinesigenic dyskinesia (PKD) with or without infantile convulsions (IC) (PKD/IC syndrome). Episodic neurologic disorders, such as epilepsy, migraine, and paroxysmal movement disorders, often coexist and are thought to have a shared channel-related etiology. To investigate further the frequency, spectrum, and phenotype of PRRT2 mutations, we analyzed this gene in 3 large series of episodic neurologic disorders with PKD/IC, episodic ataxia (EA), and hemiplegic migraine (HM).
The PRRT2 gene was sequenced in 58 family probands/sporadic individuals with PKD/IC, 182 with EA, 128 with HM, and 475 UK and 96 Asian controls.
PRRT2 genetic mutations were identified in 28 out of 58 individuals with PKD/IC (48%), 1/182 individuals with EA, and 1/128 individuals with HM. A number of loss-of-function and coding missense mutations were identified; the most common mutation found was the p.R217Pfs*8 insertion. Males were more frequently affected than females (ratio 52:32). There was a high proportion of PRRT2 mutations found in families and sporadic cases with PKD associated with migraine or HM (10 out of 28). One family had EA with HM and another large family had typical HM alone.
This work expands the phenotype of mutations in the PRRT2 gene to include the frequent occurrence of migraine and HM with PKD/IC, and the association of mutations with EA and HM and with familial HM alone. We have also extended the PRRT2 mutation type and frequency in PKD and other episodic neurologic disorders.
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.
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.
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.
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.
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.
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.
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.