Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

Modulators of Kv3 Potassium Channels Rescue the Auditory Function of Fragile X Mice.

Fragile X syndrome (FXS) is characterized by hypersensitivity to sensory stimuli, including environmental sounds. We compared the auditory brainstem response (ABR) recorded in vivo in mice lacking the gene (Fmr1-/y ) for fragile X mental retardation protein (FMRP) with that in wild-type animals. We found that ABR wave I, which represents input from the auditory nerve, is reduced in Fmr1-/y animals, but only at high sound levels. In contrast, wave IV, which represents the activity of auditory brainstem nuclei is enhanced at all sound levels, suggesting that loss of FMRP alters the central processing of auditory signals. Current-clamp recordings of neurons in the medial nucleus of the trapezoid body in the auditory brainstem revealed that, in contrast to neurons from wild-type animals, sustained depolarization triggers repetitive firing rather than a single action potential. In voltage-clamp recordings, K+ currents that activate at positive potentials ("high-threshold" K+ currents), which are required for high-frequency firing and are carried primarily by Kv3.1 channels, are elevated in Fmr1-/y mice, while K+ currents that activate near the resting potential and inhibit repetitive firing are reduced. We therefore tested the effects of AUT2 [((4-({5-[(4R)-4-ethyl-2,5-dioxo-1-imidazolidinyl]-2-pyridinyl}oxy)-2-(1-methylethyl) benzonitrile], a compound that modulates Kv3.1 channels. AUT2 reduced the high-threshold K+ current and increased the low-threshold K+ currents in neurons from Fmr1-/y animals by shifting the activation of the high-threshold current to more negative potentials. This reduced the firing rate and, in vivo, restored wave IV of the ABR. Our results from animals of both sexes suggest that the modulation of the Kv3.1 channel may have potential for the treatment of sensory hypersensitivity in patients with FXS.SIGNIFICANCE STATEMENT mRNA encoding the Kv3.1 potassium channel was one of the first described targets of the fragile X mental retardation protein (FMRP). Fragile X syndrome is caused by loss of FMRP and, in humans and mice, causes hypersensitivity to auditory stimuli. We found that components of the auditory brain response (ABR) corresponding to auditory brainstem activity are enhanced in mice lacking FMRP. This is accompanied by hyperexcitability and altered potassium currents in auditory brainstem neurons. Treatment with a drug that alters the voltage dependence of Kv3.1 channels normalizes the imbalance of potassium currents, as well as ABR responses in vivo, suggesting that such compounds may be effective in treating some symptoms of fragile X syndrome.

Physiological modulators of Kv3.1 channels adjust firing patterns of auditory brain stem neurons.

Many rapidly firing neurons, including those in the medial nucleus of the trapezoid body (MNTB) in the auditory brain stem, express "high threshold" voltage-gated Kv3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. We now describe the actions of two imidazolidinedione derivatives, AUT1 and AUT2, which modulate Kv3.1 channels. Using Chinese hamster ovary cells stably expressing rat Kv3.1 channels, we found that lower concentrations of these compounds shift the voltage of activation of Kv3.1 currents toward negative potentials, increasing currents evoked by depolarization from typical neuronal resting potentials. Single-channel recordings also showed that AUT1 shifted the open probability of Kv3.1 to more negative potentials. Higher concentrations of AUT2 also shifted inactivation to negative potentials. The effects of lower and higher concentrations could be mimicked in numerical simulations by increasing rates of activation and inactivation respectively, with no change in intrinsic voltage dependence. In brain slice recordings of mouse MNTB neurons, both AUT1 and AUT2 modulated firing rate at high rates of stimulation, a result predicted by numerical simulations. Our results suggest that pharmaceutical modulation of Kv3.1 currents represents a novel avenue for manipulation of neuronal excitability and has the potential for therapeutic benefit in the treatment of hearing disorders.

A Novel Modulator of Kv3 Potassium Channels Regulates the Firing of Parvalbumin-Positive Cortical Interneurons.

Kv3.1 and Kv3.2 high voltage-activated potassium channels, which display fast activation and deactivation kinetics, are known to make a crucial contribution to the fast-spiking phenotype of certain neurons. Pharmacological experiments show that the blockade of native Kv3 currents with low concentrations of tetraethylammonium or 4-aminopyridine impairs the expression of this firing phenotype. In particular, Kv3 channels are highly expressed by fast-spiking, parvalbumin-positive interneurons in corticolimbic brain circuits, which modulate the synchronization of cortical circuits and the generation of brain rhythms. Here, we describe a novel small molecule, (5R)-5-ethyl-3-(6-{[4-methyl-3-(methyloxy)phenyl]oxy}-3-pyridinyl)-2,4-imidazolidinedione (AUT1), which modulates Kv3.1 and Kv3.2 channels in human recombinant and rodent native neurons. AUT1 increased whole currents mediated by human Kv3.1b and Kv3.2a channels, with a concomitant leftward shift in the voltage dependence of activation. A less potent effect was observed on hKv3.3 currents. In mouse somatosensory cortex slices in vitro, AUT1 rescued the fast-spiking phenotype of parvalbumin-positive-fast-spiking interneurons following an impairment of their firing capacity by blocking a proportion of Kv3 channels with a low concentration of tetraethylammonium. Notably, AUT1 had no effect on interneuron firing when applied alone. Together, these data confirm the role played by Kv3 channels in the regulation of the firing phenotype of somatosensory interneurons and suggest that AUT1 and other Kv3 modulators could represent a new and promising therapeutic approach to the treatment of disorders associated with dysfunction of inhibitory feedback in corticolimbic circuits, such as schizophrenia.

Mechanisms contributing to central excitability changes during hearing loss.

Exposure to loud sound causes cochlear damage resulting in hearing loss and tinnitus. Tinnitus has been related to hyperactivity in the central auditory pathway occurring weeks after loud sound exposure. However, central excitability changes concomitant to hearing loss and preceding those periods of hyperactivity, remain poorly explored. Here we investigate mechanisms contributing to excitability changes in the dorsal cochlear nucleus (DCN) shortly after exposure to loud sound that produces hearing loss. We show that acoustic overexposure alters synaptic transmission originating from the auditory and the multisensory pathway within the DCN in different ways. A reduction in the number of myelinated auditory nerve fibers leads to a reduced maximal firing rate of DCN principal cells, which cannot be restored by increasing auditory nerve fiber recruitment. In contrast, a decreased membrane resistance of DCN granule cells (multisensory inputs) leads to a reduced maximal firing rate of DCN principal cells that is overcome when additional multisensory fibers are recruited. Furthermore, gain modulation by inhibitory synaptic transmission is disabled in both auditory and multisensory pathways. These cellular mechanisms that contribute to decreased cellular excitability in the central auditory pathway are likely to represent early neurobiological markers of hearing loss and may suggest interventions to delay or stop the development of hyperactivity that has been associated with tinnitus.

The binding and mechanism of a positive allosteric modulator of Kv3 channels.

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel's tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation.

Targeted therapy improves cellular dysfunction, ataxia, and seizure susceptibility in a model of a progressive myoclonus epilepsy.

The recurrent variant KCNC1-p.Arg320His causes progressive myoclonus epilepsy (EPM) type 7, defined by progressive myoclonus, epilepsy, and ataxia, and is without effective treatment. KCNC1 encodes the voltage-gated potassium channel subunit Kv3.1, specifically expressed in high-frequency-firing neurons. Variant subunits act via loss of function; hence, EPM7 pathogenesis may involve impaired excitability of Kv3.1-expressing neurons, while enhancing Kv3 activity could represent a viable therapeutic strategy. We generate a mouse model, Kcnc1-p.Arg320His/+, which recapitulates the core features of EPM7, including progressive ataxia and seizure susceptibility. Kv3.1-expressing cerebellar granule cells and neocortical parvalbumin-positive GABAergic interneurons exhibit abnormalities consistent with Kv3 channel dysfunction. A Kv3-specific positive modulator (AUT00206) selectively enhances the firing frequency of Kv3.1-expressing neurons and improves motor function and seizure susceptibility in Kcnc1-Arg320His/+ mice. This work identifies a cellular and circuit basis of dysfunction in EPM7 and demonstrates that Kv3 positive modulators such as AUT00206 have therapeutic potential for the treatment of EPM7.

Pharmacological modulation of Kv3 voltage-gated potassium channels regulates fear discrimination and expression in a response-dependent manner.

Various psychiatric diseases are characterized by aberrant cognition and emotional regulation. This includes inappropriately attributing affective salience to innocuous cues, which can be investigated using translationally relevant preclinical models of fear discrimination. Activity in the underpinning corticolimbic circuitry is governed by parvalbumin-expressing GABAergic interneurons, which also regulate fear discrimination. Kv3 voltage-gated potassium channels are highly expressed in these neurons and are important for controlling their activity, suggesting that pharmacological Kv3 modulation may regulate fear discrimination. We determined the effect of the positive Kv3 modulator AUT00206 given systemically to female rats undergoing limited or extended auditory fear discrimination training, which we have previously shown results in more discrimination or generalization, respectively, based on freezing at retrieval. We also characterized darting and other active fear-related responses. We found that limited training resulted in more discrimination based on freezing, which was unaffected by AUT00206. In contrast, extended training resulted in more generalization based on freezing and the emergence of discrimination based on darting during training and, to a lesser extent, at retrieval. Importantly, AUT00206 given before extended training had dissociable effects on fear discrimination and expression at retrieval depending on the response examined. While AUT00206 mitigated generalization without affecting expression based on freezing, it reduced expression without affecting discrimination based on darting, although darting levels were low overall. These results indicate that pharmacological Kv3 modulation regulates fear discrimination and expression in a response-dependent manner. They also raise the possibility that targeting Kv3 channels may ameliorate perturbed cognition and emotional regulation in psychiatric disease.

The mechanism of action of retigabine (ezogabine), a first-in-class K+ channel opener for the treatment of epilepsy.

The pharmacologic profile of retigabine [RTG (international nonproprietary name); ezogabine, EZG (U.S. adopted name)], is different from all currently approved antiepileptic drugs (AEDs). Its primary mechanism of action (MoA) as a positive allosteric modulator of KCNQ2-5 (K(v) 7.2-7.5) ion channels defines RTG/EZG as the first neuronal potassium (K(+)) channel opener for the treatment of epilepsy. KCNQ2-5 channels are predominantly expressed in neurons and are important determinants of cellular excitability, as indicated by the occurrence of human genetic mutations in KCNQ channels that underlie inheritable disorders including, in the case of KCNQ2/3, the syndrome of benign familial neonatal convulsions. In vitro pharmacologic studies demonstrate that the most potent action of RTG/EZG is at KCNQ2-5 channels, particularly heteromeric KCNQ2/3. Furthermore, mutagenesis and modeling studies have pinpointed the RTG/EZG binding site to a hydrophobic pocket near the channel gate, indicating how RTG/EZG can stabilize the open form of KCNQ2-5 channels; the absence of this site in KCNQ1 also provides a clear explanation for the inbuilt selectivity RTG/EZG has for potassium channels other than the KCNQ cardiac channel. KCNQ channels are active at the normal cell resting membrane potential (RMP) and contribute a continual hyperpolarizing influence that stabilizes cellular excitability. The MoA of RTG/EZG increases the number of KCNQ channels that are open at rest and also primes the cell to retort with a larger, more rapid, and more prolonged response to membrane depolarization or increased neuronal excitability. In this way, RTG/EZG amplifies this natural inhibitory force in the brain, acting like a brake to prevent the high levels of neuronal action potential burst firing (epileptiform activity) that may accompany sustained depolarizations associated with the initiation and propagation of seizures. This action to restore physiologic levels of neuronal activity is thought to underlie the efficacy of RTG/EZG as an anticonvulsant in a broad spectrum of preclinical seizure models and in placebo-controlled trials in patients with partial epilepsy. In this article, we consider the pharmacologic characteristics of RTG/EZG at the receptor, cellular, and network levels as a means of understanding the novel and efficacious MoA of this new AED as defined in both preclinical and clinical research.

The spectrum of anticonvulsant efficacy of retigabine (ezogabine) in animal models: implications for clinical use.

Retigabine [RTG (international nonproprietary name); ezogabine (EZG; U.S. adopted name)] is a first-in-class antiepileptic drug (AED) that reduces neuronal excitability by enhancing the activity of KCNQ (K(v)7) potassium (K(+)) channels. RTG/EZG has recently been approved by the European Medicines Agency and the U.S. Food and Drug Administration as adjunctive therapy in adults with partial-onset seizures. In this review we discuss the activity that RTG/EZG has demonstrated across a broad spectrum of in vitro/in vivo animal models of seizures, including generalized tonic-clonic, primary generalized (absence), and partial seizures, in addition to the compound's ability to resist and block the occurrence of seizures induced by a range of stimuli across different regions of the brain. The potency of RTG/EZG in models refractory to several conventional AEDs and the work done to assess antiepileptogenesis and neuroprotection are discussed. Studies that have evaluated the central nervous system side effects of RTG/EZG in animals are reviewed in order to compare these effects with adverse events observed in patients with epilepsy. Based on its demonstrated effect in a number of animal epilepsy models, the synergistic and additive activity of RTG/EZG with other AEDs supports its potential use in therapeutic combinations for different seizure types. The distinct mechanism of action of RTG/EZG from those of currently available AEDs, along with its broad preclinical activity, underscores the key role of KCNQ (K(v)7) K(+) channels in neuronal excitability, and further supports the potential efficacy of this unique molecule in the treatment of epilepsy.

Kv3 Channels Contribute to the Excitability of Subpopulations of Spinal Cord Neurons in Lamina VII.

Autonomic parasympathetic preganglionic neurons (PGNs) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be because of recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within Lamina VII, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphologic analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast-firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.

Reducing voltage-dependent potassium channel Kv3.4 levels ameliorates synapse loss in a mouse model of Alzheimer's disease.

Synapse loss is associated with cognitive decline in Alzheimer's disease, and owing to their plastic nature, synapses are an ideal target for therapeutic intervention. Oligomeric amyloid beta around amyloid plaques is known to contribute to synapse loss in mouse models and is associated with synapse loss in human Alzheimer's disease brain tissue, but the mechanisms leading from Aß to synapse loss remain unclear. Recent data suggest that the fast-activating and -inactivating voltage-gated potassium channel subtype 3.4 (Kv3.4) may play a role in Aß-mediated neurotoxicity. Here, we tested whether this channel could also be involved in Aß synaptotoxicity. Using adeno-associated virus and clustered regularly interspaced short palindromic repeats technology, we reduced Kv3.4 expression in neurons of the somatosensory cortex of APP/PS1 mice. These mice express human familial Alzheimer's disease-associated mutations in amyloid precursor protein and presenilin-1 and develop amyloid plaques and plaque-associated synapse loss similar to that observed in Alzheimer's disease brain. We observe that reducing Kv3.4 levels ameliorates dendritic spine loss and changes spine morphology compared to control virus. In support of translational relevance, Kv3.4 protein was observed in human Alzheimer's disease and control brain and is associated with synapses in human induced pluripotent stem cell-derived cortical neurons. We also noted morphological changes in induced pluripotent stem cell neurones challenged with human Alzheimer's disease-derived brain homogenate containing Aß but, in this in vitro model, total mRNA levels of Kv3.4 were found to be reduced, perhaps as an early compensatory mechanism for Aß-induced damage. Overall, our results suggest that approaches to reduce Kv3.4 expression and/or function in the Alzheimer's disease brain could be protective against Aß-induced synaptic alterations.

The efficacy of sodium channel blockers to prevent phencyclidine-induced cognitive dysfunction in the rat: potential for novel treatments for schizophrenia.

Sodium channel inhibition is a well precedented mechanism used to treat epilepsy and other hyperexcitability disorders. The established sodium channel blocker and broad-spectrum anticonvulsant lamotrigine is also effective in the treatment of bipolar disorder and has been evaluated in patients with schizophrenia. Double-blind placebo-controlled clinical trials found that the drug has potential to reduce cognitive symptoms of the disorder. However, because of compound-related side-effects and the need for dose titration, a conclusive evaluation of the drug's efficacy in patients with schizophrenia has not been possible. (5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-l-prolinamide (GSK2) and (2R,5R)-2-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-7-methyl-1,7-diazaspiro[4.4]nonan-6-one (GSK3) are two new structurally diverse sodium channel blockers with potent anticonvulsant activity. In this series of studies in the rat, we compared the efficacy of the two new molecules to prevent a cognitive deficit induced by the N-methyl-d-aspartic acid receptor antagonist phencyclidine (PCP) in the reversal-learning paradigm in the rat. We also explored the effects of the drugs to prevent brain activation and neurochemical effects of PCP. We found that, like lamotrigine, both GSK2 and GSK3 were able to prevent the deficit in reversal learning produced by PCP, thus confirming their potential in the treatment of cognitive symptoms of schizophrenia. However, higher doses than those required for anticonvulsant efficacy of the drugs were needed for activity in the reversal-learning model, suggesting a lower therapeutic window relative to mechanism-dependent central side effects for this indication.

Using interleaved transcranial magnetic stimulation/functional magnetic resonance imaging (fMRI) and dynamic causal modeling to understand the discrete circuit specific changes of medications: lamotrigine and valproic acid changes in motor or prefrontal effective connectivity.

The purpose of this study was to use interleaved transcranial magnetic stimulation/functional magnetic resonance imaging (TMS/fMRI) to investigate the effects of lamotrigine (LTG) and valproic acid (VPA) on effective connectivity within motor and corticolimbic circuits. In this randomized, double-blind, crossover trial, 30 healthy volunteers received either drug or placebo 3.5 h prior to interleaved TMS/fMRI. We utilized dynamic causal modeling (DCM) to assess changes in the endogenous effective connectivity of bidirectional networks in the motor-sensory system and corticolimbic circuit. Results indicate that both LTG and VPA have network-specific effects. When TMS was applied over the motor cortex, both LTG and VPA reduced TMS-specific effective connectivity between primary motor (M1) and pre-motor cortex (PMd), and between M1 and the supplementary area motor (SMA). When TMS was applied over prefrontal cortex, however, LTG alone increased TMS-specific effective connectivity between the left dorsolateral prefrontal cortex(DLPFC) and the anterior cingulate cortex (ACC). In summary, LTG and VPA inhibited effective connectivity in motor circuits, but LTG alone increased effective connectivity in prefrontal circuits. These results suggest that interleaved TMS/fMRI can assess region- and circuit-specific effects of medications or interventions.

Effect of Kv3 channel modulators on auditory temporal resolution in aged Fischer 344 rats.

AUT00063 and AUT00202 are novel pharmaceutical modulators of the Kv3 subfamily of voltage-gated K+ channels. Kv3.1 channels, which control fast firing of many central auditory neurons, have been shown to decline with age and this may contribute to age-related deficits in central auditory processing. In the present study, the effects of the two novel compounds that specifically modulate Kv3 channels on auditory temporal processing were examined in aged (19-25-month-old) and young-adult (3-5 month-old) Fischer 344 rats (F344) using a behavioral gap-prepulse inhibition (gap-PPI) paradigm. The acoustic startle response (ASR) and its inhibition induced by a gap in noise were measured before and after drug administration. Hearing thresholds in tested rats were evaluated by the auditory brainstem response (ABR). Aged F344 rats had significantly higher ABR thresholds, lower amplitudes of ASR, and weaker gap-PPI compared with young-adult rats. No influence of AUT00063 and AUT00202 administration was observed on ABR hearing thresholds in rats of both age groups. AUT00063 and AUT00202 had suppressive effect on ASR of F344 rats that was more pronounced with AUT00063. The degree of suppression depended on the dose and age of the rats. Both compounds significantly improved the gap-PPI performance in gap detection tests in aged rats. These results indicate that AUT00063 and AUT00202 may influence intrinsic firing properties of neurons in the central auditory system of aged animals and have the potential to treat aged-related hearing disorders.

Potentiation of the anticonvulsant efficacy of sodium channel inhibitors by an NK1-receptor antagonist in the rat.

PURPOSE: Many patients with epilepsy are refractory to anticonvulsant drugs or do not tolerate side effects associated with the high doses required to fully prevent seizures. Antagonists of neurokinin-1 (NK1) receptors have the potential to reduce seizure severity, although this potential has not been fully explored in animals or humans. The present study was designed to evaluate the efficacy of the NK1-receptor antagonist, vofopitant, alone and in combination with different anticonvulsant drugs. METHODS: Studies were conducted in rats using a model of generalized seizure induced by electroshock. Drug concentrations in blood and brain were determined in parallel to distinguish pharmacodynamic from pharmacokinetic interactions. RESULTS: The NK1-receptor antagonist, GR205171 (vofopitant) had no anticonvulsant efficacy by itself, but could potentiate the anticonvulsant efficacy of lamotrigine and other sodium channel blockers. However, GR205171 had no effect on the anticonvulsant potency of either valproate or gabapentin. GR205171 did not produce central nervous system (CNS) side effects at the doses tested, and it did not potentiate side effects induced by high doses of lamotrigine. The NK1-receptor inactive enantiomer of GR205171, GR226206 did not potentiate the efficacy of lamotrigine, suggesting that effects observed with GR205171 were mediated by NK1 receptors. Analysis of the dose-effect relationship for GR205171 indicated that a high (>99%) occupancy of NK1 receptors is required for effect, consistent with previous behavioral and human clinical studies with this pharmacologic class. DISCUSSION: These results suggest that there may be benefit in adding treatment with a suitable NK1-receptor antagonist to treatment with a sodium channel blocker in patients with refractory epilepsy.

Neural network dysfunction in bipolar depression: clues from the efficacy of lamotrigine.

One strategy to understand bipolar disorder is to study the mechanism of action of mood-stabilizing drugs, such as valproic acid and lithium. This approach has implicated a number of intracellular signalling elements, such as GSK3beta (glycogen synthase kinase 3beta), ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) or protein kinase C. However, lamotrigine does not seem to modulate any of these targets, which is intriguing given that its profile in the clinic differs from that of valproic acid or lithium, with greater efficacy to prevent episodes of depression than mania. The primary target of lamotrigine is the voltage-gated sodium channel, but it is unclear why inhibition of these channels might confer antidepressant efficacy. In healthy volunteers, we found that lamotrigine had a facilitatory effect on the BOLD (blood-oxygen-level-dependent) response to TMS (transcranial magnetic stimulation) of the prefrontal cortex. This effect was in contrast with an inhibitory effect of lamotrigine when TMS was applied over the motor cortex. In a follow-up study, a similar prefrontal specific facilitatory effect was observed in a larger cohort of healthy subjects, whereas valproic acid inhibited motor and prefrontal cortical TMS-induced BOLD response. In vitro, we found that lamotrigine (3-10 microM) enhanced the power of gamma frequency network oscillations induced by kainic acid in the rat hippocampus, an effect that was not observed with valproic acid (100 microM). These data suggest that lamotrigine has a positive effect on corticolimbic network function that may differentiate it from other mood stabilizers. The results are also consistent with the notion of corticolimbic network dysfunction in bipolar disorder.

Lamotrigine and valproic acid have different effects on motorcortical neuronal excitability.

To investigate different cortical effects of lamotrigine and valproic acid, 30 paid healthy adult men were given, in a randomized/blinded fashion on three separate days (separated by a week), either a single dose of lamotrigine 325 mg, or a single dose of valproic acid 1,250 mg, or placebo. Resting motor threshold (RMT), cortical silent period (CSP) and motor evoked potential recruitment curves (RC) were assessed at baseline and 3 h after administration of each medication (or placebo). Lamotrigine caused a significant increase (63.32 vs. 69.25) in the RMT, compared with an insignificant increase following valproic acid (62.50 vs. 63.35), and a decrease (62.60 vs. 62.36) following placebo (F (2,26) = 18.58, P < 0.0001). No significant difference in CSP was found between placebo and drugs (F (2,26) = 0.119, P > 0.05). RCs were significantly suppressed by lamotrigine (t = 2.07, P < 0.05) and enhanced by valproic acid (t = 2.39, P < 0.05). Lamotrigine and valproic acid have different effects on cortical neuronal excitability as demonstrated by TMS.

Automated patch clamping using the QPatch.

Whole-cell voltage clamp electrophysiology using glass patch pipettes (1) is regarded as the gold standard for measurement of compound activity on ion channels. Despite the high quality of the data generated by this method, in its traditional format, patch clamping has limited use in drug screening due to very low throughput. Over the years, developments in microfabrication have driven the development of planar, multi-aperture technologies that are suitable for parallel, automated patch recording techniques. Here we present detailed methods for two common applications of the planar patch technology using one of the commercially available instruments. The results demonstrate (a) the high quality of whole-cell recordings obtainable from cell lines expressing human Nav1.2 or hERG ion channels, (b) the advantages of the methodology for increasing throughput, and (c) examples of how these assays support ion channel drug discovery.

Why Is There No Cure for Tinnitus?

Tinnitus is unusual for such a common symptom in that there are few treatment options and those that are available are aimed at reducing the impact rather than specifically addressing the tinnitus percept. In particular, there is no drug recommended specifically for the management of tinnitus. Whilst some of the currently available interventions are effective at improving quality of life and reducing tinnitus-associated psychological distress, most show little if any effect on the primary symptom of subjective tinnitus loudness. Studies of the delivery of tinnitus services have demonstrated considerable end-user dissatisfaction and a marked disconnect between the aims of healthcare providers and those of tinnitus patients: patients want their tinnitus loudness reduced and would prefer a pharmacological solution over other modalities. Several studies have shown that tinnitus confers a significant financial burden on healthcare systems and an even greater economic impact on society as a whole. Market research has demonstrated a strong commercial opportunity for an effective pharmacological treatment for tinnitus, but the amount of tinnitus research and financial investment is small compared to other chronic health conditions. There is no single reason for this situation, but rather a series of impediments: tinnitus prevalence is unclear with published figures varying from 5.1 to 42.7%; there is a lack of a clear tinnitus definition and there are multiple subtypes of tinnitus, potentially requiring different treatments; there is a dearth of biomarkers and objective measures for tinnitus; treatment research is associated with a very large placebo effect; the pathophysiology of tinnitus is unclear; animal models are available but research in animals frequently fails to correlate with human studies; there is no clear definition of what constitutes meaningful change or "cure"; the pharmaceutical industry cannot see a clear pathway to distribute their products as many tinnitus clinicians are non-prescribing audiologists. To try and clarify this situation, highlight important areas for research and prevent wasteful duplication of effort, the British Tinnitus Association (BTA) has developed a Map of Tinnitus. This is a repository of evidence-based tinnitus knowledge, designed to be free to access, intuitive, easy to use, adaptable and expandable.