Modulating Behavior in C. elegans Using Electroshock and Antiepileptic Drugs

The microscopic nematode Caenorhabditis elegans has emerged as a valuable model for understanding the molecular and cellular basis of neurological disorders. The worm offers important physiological similarities to mammalian models such as conserved neuron morphology, ion channels, and neurotransmitters. While a wide-array of behavioral assays are available in C. elegans, an assay for electroshock/electroconvulsion remains absent. Here, we have developed a quantitative behavioral method to assess the locomotor response following electric shock in C. elegans. Electric shock impairs normal locomotion, and induces paralysis and muscle twitching; after a brief recovery period, shocked animals resume normal locomotion. We tested electric shock responses in loss-of-function mutants for unc-25, which encodes the GABA biosynthetic enzyme GAD, and unc-49, which encodes the GABAA receptor. unc-25 and unc-49 mutants have decreased inhibitory GABAergic transmission to muscles, and take significantly more time to recover normal locomotion following electric shock compared to wild-type. Importantly, increased sensitivity of unc-25 and unc-49 mutants to electric shock is rescued by treatment with antiepileptic drugs, such as retigabine. Additionally, we show that pentylenetetrazol (PTZ), a GABAA receptor antagonist and proconvulsant in mammalian and C. elegans seizure models, increases susceptibility of worms to electric shock.

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title: Use of Caenorhabditis elegans as a model to study Alzheimer’s disease and other neurodegenerative diseases
abstract: Advances in research and technology has increased our quality of life, allowed us to combat diseases, and achieve increased longevity. Unfortunately, increased longevity is accompanied by a rise in the incidences of age-related diseases such as Alzheimer’s disease (AD). AD is the sixth leading cause of death, and one of the leading causes of dementia amongst the aged population in the USA. It is a progressive neurodegenerative disorder, characterized by the prevalence of extracellular Aβ plaques and intracellular neurofibrillary tangles, derived from the proteolysis of the amyloid precursor protein (APP) and the hyperphosphorylation of microtubule-associated protein tau, respectively. Despite years of extensive research, the molecular mechanisms that underlie the pathology of AD remain unclear. Model organisms, such as the nematode, Caenorhabditis elegans, present a complementary approach to addressing these questions. C. elegans has many advantages as a model system to study AD and other neurodegenerative diseases. Like their mammalian counterparts, they have complex biochemical pathways, most of which are conserved. Genes in which mutations are correlated with AD have counterparts in C. elegans, including an APP-related gene, apl-1, a tau homolog, ptl-1, and presenilin homologs, such as sel-12 and hop-1. Since the neuronal connectivity in C. elegans has already been established, C. elegans is also advantageous in modeling learning and memory impairments seen during AD. This article addresses the insights C. elegans provide in studying AD and other neurodegenerative diseases. Additionally, we explore the advantages and drawbacks associated with using this model.

title: Using C. elegans to Decipher the Cellular and Molecular Mechanisms Underlying Neurodevelopmental Disorders
abstract: Neurodevelopmental disorders such as epilepsy, intellectual disability (ID), and autism spectrum disorders (ASDs) occur in over 2 % of the population, as the result of genetic mutations, environmental factors, or combination of both. In the last years, use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. Nevertheless, following association of novel genes with a given disease, interpretation of findings is often difficult due to lack of information on gene function and effect of a given mutation in the corresponding protein. This brings the need to validate genetic associations from a functional perspective in model systems in a relatively fast but effective manner. In this context, the small nematode, Caenorhabditis elegans, presents a good compromise between the simplicity of cell models and the complexity of rodent nervous systems. In this article, we review the features that make C. elegans a good model for the study of neurodevelopmental diseases. We discuss its nervous system architecture and function as well as the molecular basis of behaviors that seem important in the context of different neurodevelopmental disorders. We review methodologies used to assess memory, learning, and social behavior as well as susceptibility to seizures in this organism. We will also discuss technological progresses applied in C. elegans neurobiology research, such as use of microfluidics and optogenetic tools. Finally, we will present some interesting examples of the functional analysis of genes associated with human neurodevelopmental disorders and how we can move from genes to therapies using this simple model organism.

title: A Predictable Worm: Application of Caenorhabditis elegans for Mechanistic Investigation of Movement Disorders
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title: An RNAi Screen Identifies Genes that Regulate GABA Synapses
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title: One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction
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title: The structure of the nervous system of the nematode Caenorhabditis elegans.
abstract: The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.

title: Antifungal Chemical Compounds Identified Using a C. elegans Pathogenicity Assay
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title: Functional genomic analysis of C. elegans chromosome I by systematic RNA interference
abstract: Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster , and it will soon be known for humans. However, biological function has been assigned to only a small proportion of the predicted genes in any animal. Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C. elegans chromosome I by feeding worms with bacteria that express double-stranded RNA. We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378. Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function. In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation. We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.

title: Genome-wide RNAi screening in Caenorhabditis elegans.
abstract: In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.

title: A small-molecule screen in C. elegans yields a new calcium channel antagonist
abstract: Small-molecule inhibitors of protein function are powerful tools for biological analysis and can lead to the development of new drugs. However, a major bottleneck in generating useful small-molecule tools is target identification. Here we show that Caenorhabditis elegans can provide a platform for both the discovery of new bioactive compounds and target identification. We screened 14,100 small molecules for bioactivity in wild-type worms and identified 308 compounds that induce a variety of phenotypes. One compound that we named nemadipine-A induces marked defects in morphology and egg-laying. Nemadipine-A resembles a class of widely prescribed anti-hypertension drugs called the 1,4-dihydropyridines (DHPs) that antagonize the α1-subunit of L-type calcium channels. Through a genetic suppressor screen, we identified egl-19 as the sole candidate target of nemadipine-A, a conclusion that is supported by several additional lines of evidence. egl-19 encodes the only L-type calcium channel α1-subunit in the C. elegans genome. We show that nemadipine-A can also antagonize vertebrate L-type calcium channels, demonstrating that worms and vertebrates share the orthologous protein target. Conversely, FDA-approved DHPs fail to elicit robust phenotypes, making nemadipine-A a unique tool to screen for genetic interactions with this important class of drugs. Finally, we demonstrate the utility of nemadipine-A by using it to reveal redundancy among three calcium channels in the egg-laying circuit. Our study demonstrates that C. elegans enables rapid identification of new small-molecule tools and their targets.

title: Genetic interactions among cortical malformation genes that influence susceptibility to convulsions in C. elegans

abstract: Epilepsy is estimated to affect 1-2% of the world population, yet remains poorly understood at a molecular level. We have previously established the roundworm Caenorhabditis elegans as a model for investigating genetic susceptibilities to seizure-like convulsions in vivo. Here we investigate the behavioral consequences of decreasing the activity of nematode gene homologs within the LIS1 pathway that are associated with a human cortical malformation termed lissencephaly. Bioinformatic analysis revealed the nud-2 gene, encoding the worm homolog of mammalian effectors of LIS1, termed NDE1 and NDEL1. Phenotypic analysis of animals targeted by RNA interference (RNAi) was performed using a pentylenetetrazole (PTZ) exposure paradigm to induce convulsions. Worms depleted for LIS1 pathway components (NUD-1, NUD-2, DHC-1, CDK-5, and CDKA-1) exhibited significant convulsions following PTZ and RNAi treatment. Strains harboring fluorescent markers for GABAergic neuronal architecture and synaptic vesicle trafficking were employed to discern putative mechanisms accounting for observed convulsion behaviors. We found that depletion of LIS1 pathway components resulted in defective GABA synaptic vesicle trafficking. We also utilized combinations of specific genetic backgrounds to create a sensitized state for convulsion susceptibility and discovered that convulsion effects were significantly enhanced when LIS-1 and other pathway components were compromised within the same animals. Thus, interactions among gene products with LIS-1 may mediate intrinsic thresholds of neuronal synchrony.

title: Epileptic-like convulsions associated with LIS-1 in the cytoskeletal control of neurotransmitter signaling in Caenorhabditis elegans.
abstract: Cortical malformations are a collection of disorders affecting brain development. Mutations in the LIS1 gene lead to a disorganized and smooth cerebral cortex caused by failure in neuronal migration. Among the clinical consequences of lissencephaly are mental retardation and intractable epilepsy. It remains unclear whether the seizures result from aberrant neuronal placement, disruption of intrinsic properties of neurons, or both. The nematode Caenorhabditis elegans offers an opportunity to study such convulsions in a simple animal with a defined nervous system. Here we show that convulsions mimicking epilepsy can be induced by a mutation in a C. elegans lis-1 allele (pnm-1), in combination with a chemical antagonist of gamma-aminobutyric acid (GABA) neurotransmitter signaling. Identical convulsions were obtained using C. elegans mutants defective in GABA transmission, whereas none of these mutants or the antagonist alone caused convulsions, indicating a threshold was exceeded in response to this combination. Crosses between pnm-1 and fluorescent marker strains designed to exclusively illuminate either the processes of GABAergic neurons or synaptic vesicles surprisingly showed no deviations in neuronal architecture. Instead, presynaptic defects in GABAergic vesicle distribution were clearly evident and could be phenocopied by RNAi directed against cytoplasmic dynein, a known LIS1 interactor. Furthermore, mutations in UNC-104, a neuronal-specific kinesin, and SNB-1, a synaptic vesicle-associated protein termed synaptobrevin, exhibit similar convulsion phenotypes following chemical induction. Taken together, these studies establish C. elegans as a system to investigate subtle cytoskeletal mechanisms regulating intrinsic neuronal activity and suggest that it may be possible to dissociate the epileptic consequences of lissencephaly from the more phenotypically overt cortical defects associated with neuronal migration.

title: Inappropriate Neural Activity during a Sensitive Period in Embryogenesis Results in Persistent Seizure-like Behavior
abstract: Maturation of neural circuits requires activity-dependent processes that underpin the emergence of appropriate behavior in the adult. It has been proposed that disruption of these events, during specific critical periods when they exert maximal influence, may lead to neurodevelopmental diseases, including epilepsy [1, 2, 3]. However, complexity of neurocircuitry, coupled with the lack of information on network formation in mammals, makes it difficult to directly investigate this hypothesis. Alternative models, including the fruit fly Drosophila melanogaster, show remarkable similarities between experimental seizure-like activity and clinical phenotypes [4, 5, 6]. In particular, a group of flies, termed bang-sensitive (bs) mutants have been extensively used to investigate the pathophysiological mechanisms underlying seizure [7, 8, 9, 10, 11, 12]. Seizure phenotype can be measured in larval stages using an electroshock assay, and this behavior in bs mutants is dramatically reduced following ingestion of typical anti-epileptic drugs (AEDs; ). In this study we describe a critical period of embryonic development in Drosophila during which manipulation of neural activity is sufficient to significantly influence seizure behavior at postembryonic stages. We show that inhibition of elevated activity, characteristic of bs seizure models, during the critical period is sufficient to suppress seizure. By contrast, increasing neuronal excitation during the same period in wild-type (WT) is sufficient to permanently induce a seizure behavior. Further, we show that induction of seizure in WT correlates with functional alteration of motoneuron inputs that is a characteristic of bs mutants. Induction of seizure is rescued by prior administration of AEDs, opening a new perspective for early drug intervention in the treatment of genetic epilepsy.

title: Electroconvulsive Seizure Behavior in Drosophila: Analysis of the Physiological Repertoire Underlying a Stereotyped Action Pattern in Bang-Sensitive Mutants
abstract: Drosophila bang-sensitive mutants display a remarkable stereotyped behavioral sequence during mechanical disturbances. This seizure repertoire consists of initial and delayed bouts of spasm interposed with paralysis and followed by recovery of activity and a period of refractoriness to further stimulation. Electroconvulsive stimuli across the brain induced a similar seizure behavior in tethered flies, in which corresponding electrophysiological events could be readily recorded in indirect flight muscles [dorsal longitudinal muscles (DLMs)] of the giant fiber (GF) pathway. The DLM physiological repertoire consisted of initial and delayed discharges (IDs and DDs), a response failure and recovery, followed by a refractory period. Interestingly, wild-type flies also displayed the same electroconvulsive repertoire, albeit inducible only at higher stimulus intensities and with briefer expression. The DLM repertoire presumably originated from activities of distinct neural circuits subserving normal function and reflected the general sequence of excitation and depression of the nervous system as a whole, as shown by simultaneous recordings along the different body axes. The well characterized GF pathway facilitated localization of circuits responsible for response failure and ID and DD motor patterns by surgical manipulations, recording-stimulating site analysis, and genetic mosaic studies. A flight pattern generator is most likely the major contributor to shaping the DD pattern, with modifications by active integration of individual motor neurons and associated interneurons. The robust electroconvulsive repertoire of DLMs provides a convenient window for further genetic analysis of the interacting neural mechanisms underlying a stereotyped action pattern inDrosophila, which shows striking parallels with aspects of seizure in mammalian species.

title: Increased persistent Na+ current contributes to seizure in the slamdance bang-sensitive Drosophila mutant.
abstract: There is clinical need to extend the understanding of epilepsy and to find novel approaches to treat this condition. Bang-sensitive (bs) Drosophila mutants, which exhibit reduced thresholds for seizure, offer an attractive possibility to combine tractable genetics, electrophysiology, and high-throughput screening. However, despite these advantages, the precise electrophysiological aberrations that contribute to seizure have not been identified in any bs mutant. Because of this, the applicability of Drosophila as a preclinical model has not yet been established. In this study, we show that electroshock of bs slamdance (sda) larvae was sufficient to induce extended seizure-like episodes. Whole cell voltage-clamp recordings from identified motoneurons (termed aCC and RP2) showed synaptic currents that were greatly increased in both amplitude and duration. Current-clamp recordings indicated that these inputs produced longer-lived plateau depolarizations and increased action potential firing in these cells. An analysis of voltage-gated currents in these motoneurons, in both first and third instar larvae, revealed a consistently increased persistent Na(+) current (I(Nap)) and a reduced Ca(2+) current in first instar larvae, which appeared normal in older third instar larvae. That increased I(Nap) may contribute to seizure-like activity is indicated by the observation that feeding sda larvae the antiepileptic drug phenytoin, which was sufficient to reduce I(Nap), rescued both seizure-like episode duration and synaptic excitation of motoneurons. In contrast, feeding of either anemone toxin, a drug that preferentially increases I(Nap), or phenytoin to wild-type larvae was sufficient to induce a bs behavioral phenotype. Finally, we show that feeding of phenytoin to gravid sda females was sufficient to both reduce I(Nap) and synaptic currents and rescue the bs phenotype in their larval progeny, indicating that a heightened predisposition to seizure may arise as a consequence of abnormal embryonic neural development.

title: Molecular targets versus models for new antiepileptic drug discovery
abstract: Animal models have played a key role in the discovery and characterization of all marketed antiepileptic drugs (AED). The conventional wisdom is that the standard animal screening models are becoming obsolete because they fail to identify compounds that act in mechanistically new ways and as a result do not offer therapeutic advantages over presently available agents. In fact, far from only detecting me-too drugs, the models often uncover compounds with distinctive profiles of activity in various types of epilepsy and in addition have unexpected efficacy in non-epilepsy conditions, such as neuropathic pain, bipolar disorder, and migraine. Moreover, the animal models-because they are unbiased with respect to mechanism-provide an opportunity to uncover drugs that act in new ways and through new targets, such as alpha2delta and SV2A. In vitro testing is not likely to replace screening in animal models because in vitro systems cannot model the specific pharmacodynamic actions required for seizure protection, and do not assess bioavailability and brain accessibility.

title: Properties of maximal seizures, and their alteration by anticonvulant drugs and other agents.
abstract: DESPITE the high incidence of convulsive disorders and widespread use of shock therapy in the major psychoses, relatively few investigations have dealt with the physiological properties of experimental seizures (1, 2, 3, 10, 12, 14, 16, 17). Convulsive threshold has received considerable attention, particularly in the testing of anticonvulsant drugs (11, 13, 15, 18, 21, and others), but threshold is only one of many properties which might be examined in any excitable system. Furthermore, measurements of threshold are of but limited value in the detection of antiepileptic potency (5, 6, 7, 8, 19, 20). For example, diphenylhydantoin, which has been found superior to phenobarbital in the treatment of psychomotor epilepsy and of comparable value in grand mal, is relatively ineffective in raising the threshold either for metrazol or electroshock seizures in laboratory animals. Therefore we have undertaken a series of studies on other properties of experimental seizures (7, 8, 19, 20, 23). Several new techniques for the testing of anticonvulsant drugs have already evolved from these studies (7, 8, 20, 22). The technique which to date has shown the best correlation with clinical antiepileptic efficacy is based upon the ability of certain drugs to alter the character of major seizures produced by supramaximal electroshock stimulation. The present report deals with some elementary properties of maximal seizures in normal animals, and with the effects of anticonvulsant drugs upon these properties.

title: Seizures Induced by Pentylenetetrazole in the Adult Zebrafish: A Detailed Behavioral Characterization
abstract: Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.

title: Retigabine: chemical synthesis to clinical application.
abstract: Retigabine [D23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl)carbamic acid ethyl ester] is an antiepileptic drug with a recently described novel mechanism of action that involves opening of neuronal K(V)7.2-7.5 (formerly KCNQ2-5) voltage-activated K(+) channels. These channels (primarily K(V)7.2/7.3) enable generation of the M-current, a subthreshold K(+) current that serves to stabilize the membrane potential and control neuronal excitability. In this regard, retigabine has been shown to have a broad-spectrum of activity in animal models of electrically-induced (amygdala-kindling, maximal electroshock) and chemically-induced (pentylenetetrazole, picrotoxin, NMDA) epileptic seizures. These encouraging results suggest that retigabine may also prove useful in the treatment of other diseases associated with neuronal hyperexcitability. Neuropathic pain conditions are characterized by pathological changes in sensory pathways, which favor action potential generation and enhanced pain transmission. Although sometimes difficult to treat with conventional analgesics, antiepileptics can relieve some symptoms of neuropathic pain. A number of recent studies have reported that retigabine can relieve pain-like behaviors (hyperalgesia and allodynia) in animal models of neuropathic pain. Neuronal activation within several key structures within the CNS can also be observed in various animal models of anxiety. Moreover, amygdala-kindled rats, which have a lowered threshold for neuronal activation, also display enhanced anxiety-like responses. Retigabine dose-dependently reduces unconditioned anxiety-like behaviors when assessed in the mouse marble burying test and zero maze. Early clinical studies have indicated that retigabine is rapidly absorbed and distributed, and is resistant to first pass metabolism. Tolerability is good in humans when titrated up to its therapeutic dose range (600-1200 mg/day). No tolerance, dependence or withdrawal potential has been reported, although adverse effects can include mild dizziness, headache, nausea and somnolence. Thus, retigabine may prove to be useful in the treatment of a diverse range of disease states in which neuronal hyperexcitability is a common underlying factor.

title: Pharmacological and Therapeutic Properties of Valproate
abstract: Thirty-five years since its introduction into clinical use, valproate (valproic acid) has become the most widely prescribed antiepileptic drug (AED) worldwide. Its pharmacological effects involve a variety of mechanisms, including increased γ-aminobutyric acid (GABA)-ergic transmission, reduced release and/or effects of excitatory amino acids, blockade of voltage-gated sodium channels and modulation of dopaminergic and serotoninergic transmission.Valproate is available in different dosage forms for parenteral and oral use. All available oral formulations are almost completely bioavailable, but they differ in dissolution characteristics and absorption rates. In particular, sustained-release formulations are available that minimise fluctuations in serum drug concentrations during a dosing interval and can therefore be given once or twice daily.Valproic acid is about 90% bound to plasma proteins, and the degree of binding decreases with increasing drug concentration within the clinically occurring range. Valproic acid is extensively metabolised by microsomal glucuronide conjugation, mitochondrial β-oxidation and cytochrome P450-dependent ω-, (ω-1)- and (ω-2)-oxidation. The elimination half-life is in the order of 9 to 18 hours, but shorter values (5 to 12 hours) are observed in patients comedicated with enzyme-inducing agents such as phenytoin, carbamazepine and barbiturates. Valproate itself is devoid of enzyme-inducing properties, but it has the potential of inhibiting drug metabolism and can increase by this mechanism the plasma concentrations of certain coadministered drugs, including phenobarbital (phenobarbitone), lamotrigine and zidovudine.Valproate is a broad spectrum AED, being effective against all seizure types. In patients with newly diagnosed partial seizures (with or without secondary generalisation) and/or primarily generalised tonic-clonic seizures, the efficacy of valproate is comparable to that of phenytoin, carbamazepine and phenobarbital, although in most comparative trials the tolerability of phenobarbital was inferior to that of the other drugs. Valproate is generally regarded as a first-choice agent for most forms of idiopathic and symptomatic generalised epilepsies. Many of these syndromes are associated with multiple seizure types, including tonic-clonic, myoclonic and absence seizures, and prescription of a broad-spectrum drug such as valproate has clear advantages in this situation. A number of reports have also suggested that intravenous valproate could be of value in the treatment of convulsive and nonconvulsive status epilepticus, but further studies are required to establish in more detail the role of the drug in this indication.The most commonly reported adverse effects of valproate include gastrointestinal disturbances, tremor and bodyweight gain. Other notable adverse effects include encephalopathy symptoms (at times associated with hyperammonaemia), platelet disorders, pancreatitis, liver toxicity (with an overall incidence of 1 in 20 000, but a frequency as high as 1 in 600 or 1 in 800 in high-risk groups such as infants below 2 years of age receiving anticonvulsant polytherapy) and teratogenicity, including a 1 to 3% risk of neural tube defects. Some studies have also suggested that menstrual disorders and certain clinical, ultrasound or endocrine manifestations of reproductive system disorders, including polycystic ovary syndrome, may be more common in women treated with valproate than in those treated with other AEDs. However, the precise relevance of the latter findings remains to be evaluated in large, prospective, randomised studies.

title: Baccoside A suppresses epileptic-like seizure/convulsion in Caenorhabditis elegans
abstract: The 1 mm long Caenorhabditis elegans is one of the prime research tools to study different human neurodegenerative diseases. We have considered the case in which increase in the surrounding temperature of this multicellular model leads to abnormal bursts of neuronal cells that can be linked to seizure or convulsion. The induction of such seizure/convulsion mechanism was done by gradually increasing the temperature with 1x buffer (100 mM NaCl, 50 mM MgCl(2)) in adult C. elegans. In the present experiment it is demonstrated that Baccoside A can significantly reduce the seizure/convulsion in C. elegans at higher temperatures (26-28+/-1 degrees C). Furthermore, in T-type Ca(2+) channel cca-1 mutant worms, no convulsion was recorded. Our experimental results suggest that plant molecules from Bacopa monnieri may be useful in suppressing the seizure/convulsion in worms.

title: Cholinergic receptor mutants of the nematode Caenorhabditis elegans
abstract: Potential acetylcholine receptor (AChR) mutants of the nematode are selectable by resistance to the neurotoxic drug levamisole, a probable cholinergic agonist. To determine which mutants may have achieved resistance through loss of levamisole receptor function, we have assayed mutant extracts for specific 3H-meta-aminolevamisole binding activity in the presence and absence of mecamylamine. We find that mutants in 3 of the 7 genes associated with extreme levamisole resistance are obviously deficient in saturable specific 3H-meta- aminolevamisole binding activity. Mutants of the 4 other genes have abnormal binding activities that fail to undergo the apparent allosteric activation of saturable specific 3H-meta-aminolevamisole binding activity caused by mecamylamine. Thus, all 7 genes appear to be required to produce a fully functional levamisole receptor. Mutants of several other genes associated only with partial resistance to levamisole have at least grossly normal receptor binding activities.

title: Motoneurons Dedicated to Either Forward or Backward Locomotion in the Nematode Caenorhabditis elegans
abstract: Multifunctional motoneurons and muscles, which are active during forward and backward locomotion are ubiquitous in animal models. However, studies in the nematode Caenorhabditis elegans suggest that some locomotor motoneurons are necessary only for forward locomotion (dorsal B-motoneurons, DB), while others (dorsal A-motoneurons, DA) are necessary only for backward locomotion. We tested this hypothesis directly by recording the activity of these motoneurons during semirestrained locomotion. For this purpose, we used epifluorescence imaging of the genetically encoded calcium sensor cameleon, expressed in specific motoneurons, while monitoring locomotor behavior through the microscope condenser using a second camera. We found that ventral and dorsal B-motoneurons (DB and VB) were coactive during forward locomotion while ventral A-motoneurons (VA) were only active during backward locomotion. The signals we recorded correlated with the direction of locomotion but not with the faster undulatory cycles. To our knowledge, these are the first recordings of motoneuron activity in C. elegans and the only direction-dedicated motoneurons described to date.

title: An Imbalancing Act: Gap Junctions Reduce the Backward Motor Circuit Activity to Bias C. elegans for Forward Locomotion
abstract: A neural network can sustain and switch between different activity patterns to execute multiple behaviors. By monitoring the decision making for directional locomotion through motor circuit calcium imaging in behaving Caenorhabditis elegans (C. elegans), we reveal that C. elegans determines the directionality of movements by establishing an imbalanced output between the forward and backward motor circuits and that it alters directions by switching between these imbalanced states. We further demonstrate that premotor interneurons modulate endogenous motoneuron activity to establish the output imbalance. Specifically, the UNC-7 and UNC-9 innexin-dependent premotor interneuron-motoneuron coupling prevents a balanced output state that leads to movements without directionality. Moreover, they act as shunts to decrease the backward-circuit activity, establishing a persistent bias for the high forward-circuit output state that results in the inherent preference of C. elegans for forward locomotion. This study demonstrates that imbalanced motoneuron activity underlies directional movement and establishes gap junctions as critical modulators of the properties and outputs of neural circuits.

title: An Image-Free Opto-Mechanical System for Creating Virtual Environments and Imaging Neuronal Activity in Freely Moving Caenorhabditis elegans
abstract: Non-invasive recording in untethered animals is arguably the ultimate step in the analysis of neuronal function, but such recordings remain elusive. To address this problem, we devised a system that tracks neuron-sized fluorescent targets in real time. The system can be used to create virtual environments by optogenetic activation of sensory neurons, or to image activity in identified neurons at high magnification. By recording activity in neurons of freely moving C. elegans, we tested the long-standing hypothesis that forward and reverse locomotion are generated by distinct neuronal circuits. Surprisingly, we found motor neurons that are active during both types of locomotion, suggesting a new model of locomotion control in C. elegans. These results emphasize the importance of recording neuronal activity in freely moving animals and significantly expand the potential of imaging techniques by providing a mean to stabilize fluorescent targets.

title: Valproic acid ameliorates C. elegans dopaminergic neurodegeneration with implications for ERK-MAPK signaling
abstract: Parkinson's disease (PD) is a currently incurable neurodegenerative disorder that affects the aging population. The loss of dopaminergic neurons in the substantia nigra is one of the pathological features of PD. The precise causes of PD remain unresolved but evidence supports both environmental and genetic contributions. Current efforts for the treatment of PD are directed toward the discovery of compounds that show promise in impeding age-dependent neurodegeneration in PD patients. Alpha-synuclein (α-Syn) is a human protein that is mutated in specific populations of patients with familial PD. Overexpression of α-Syn in animal models of PD replicates key symptoms of PD, including neurodegeneration. Here, we use the nematode Caenorhabditis elegans as a model system, whereby α-Syn toxicity causes dopaminergic neurodegeneration, to test the capacity of valproic acid (VA) to protect neurons. The results of our study showed that treatment of nematodes with moderate concentrations of VA significantly protects dopaminergic neurons against α-Syn toxicity. Consistent with previously established knowledge related to the mechanistic action of VA in the cell, we showed through genetic analysis that the neuroprotection conferred by VA is inhibited by cell-specific depletion of the C. elegans ortholog of the MAP extracellular signal-regulated kinase (ERK), MPK-1, in the dopaminergic neurons. These findings suggest that VA may exert its neuroprotective effect via ERK-MAPK, or alternately could act with MAPK signaling to additively provide dopaminergic neuroprotection.

title: Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay
abstract: Caenorhabditis elegans has emerged as a powerful model system for studying the biology of the synapse. Here we describe a widely used assay for synaptic transmission at the C. elegans neuromuscular junction. This protocol monitors the sensitivity of C. elegans to the paralyzing affects of an acetylcholinesterase inhibitor, aldicarb. Briefly, adult worms are incubated in the presence of aldicarb and scored for the time-course of aldicarb-induced paralysis. Animals harboring mutations in genes that affect synaptic transmission generally exhibit a change in their sensitivity to aldicarb (either increased sensitivity for enhancements in synaptic transmission or decreased sensitivity for blockage in synaptic transmission). This technique provides a simple assay for the accurate comparative analysis of synaptic transmission in multiple C. elegans strains. The protocol described can be performed relatively quickly and is a practical alternative to other techniques used to study synaptic transmission. This protocol can also be modified to follow the paralytic effects with other pharmacological reagents. The assay can be performed in about 3-6 hours depending on the severity of synaptic transmission defects.

title: SV2A and SV2B Function as Redundant Ca2+ Regulators in Neurotransmitter Release
abstract: SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor isoform (SV2C) that resemble transporter proteins. We now show that SV2B knockout mice are phenotypically normal while SV2A- and SV2A/SV2B double knockout mice exhibit severe seizures and die postnatally. In electrophysiological recordings from cultured hippocampal neurons, SV2A- or SV2B-deficient cells exhibited no detectable abnormalities. Neurons lacking both SV2 isoforms, however, experienced sustained increases in Ca2+-dependent synaptic transmission when two or more action potentials were triggered in succession. These increases could be reversed by EGTA-AM. Our data suggest that without SV2 proteins, presynaptic Ca2+ accumulation during consecutive action potentials causes abnormal increases in neurotransmitter release that destabilize synaptic circuits and induce epilepsy.

title: The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam.
abstract: Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of approximately 90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.

title: The new anticonvulsant retigabine (D-23129) acts as an opener of K+ channels in neuronal cells.
abstract: The patch-clamp technique was used to measure currents passing through K+ channels in neuronal cell preparations. Retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) activated a K+ conductance in slightly depolarized NG108-15 neuronal cells in a dose-dependent manner (0.1-10 microM). At the K+ reversal potential, no current could be elicited and in hyperpolarized cells the current was reversed. A similar current was elicited in primary cultures of mouse cortical neurones and in differentiated hNT cells, a cell line derived from human neuronal cells. At higher concentrations, retigabine also partially blocked voltage activated K+ currents. None of the tested anticonvulsants, phenytoin, carbamazepine and valproate and none of the K+ channel openers cromakalim, diazoxide and pinacidil exerted a similar effect. The current was not affected by the K+ channel blocker glibenclamide (10 microM) but was fully blocked by application of Ba2+ (10.8 mM). Exchange of K+ with cesium in the intracellular space also fully abolished the current. It can be expected that the K+ channel opening effect contributes to the anticonvulsant activity of retigabine.

title: KCNQ-like Potassium Channels in Caenorhabditis elegans
abstract: The human KCNQ gene family encodes potassium channels linked to several genetic syndromes including neonatal epilepsy, cardiac arrhythmia, and progressive deafness. KCNQ channels form M-type potassium channels, which are critical regulators of neuronal excitability that mediate autonomic responses, pain, and higher brain function. Fundamental mechanisms of the normal and abnormal cellular roles for these channels may be gained from their study in simple model organisms. Here we report that a multigene family of KCNQ-like channels is present in the nematode, Caenorhabditis elegans. We show that many aspects of the functional properties, tissue expression pattern, and modulation of these C. elegans channels are conserved, including suppression by the M1 muscarinic receptor. We also describe a conserved mechanism of modulation by diacylglycerol for a subset of C. elegans and vertebrate KCNQ/KQT channels, which is dependent upon the carboxyl-terminal domains of channel subunits and activated protein kinase C.

title: Drosophila KCNQ Channel Displays Evolutionarily Conserved Electrophysiology and Pharmacology with Mammalian KCNQ Channels
abstract: Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac IKs current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50–60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K+ current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies.

title: The effects of D-23129, a new experimental anticonvulsant drug, on neurotransmitter amino acids in the rat hippocampus in vitro
abstract: D-23129 [N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid ethyl ester] and D-20443 (dihydrochloride of D-23129) are promising anticonvulsant compounds with a broad spectrum activity in animal models of epilepsy. Their effects on de novo synthesis of excitatory (glutamate and aspartate) and inhibitory (GABA) amino acids were studied in rat hippocampal slices. Like phenytoin, carbamazepine, lamotrigine, losigamone, U54494A, and flupirtine, D-23129 and D-20443 were effective in preventing the effects of a chemoconvulsant, 4-aminopyridine, on de novo synthesis of the three amino acids. However, unlike the other compounds, D-23129 and D-20443 also preferentially increased the concentrations of newly synthesized GABA. Their effect on the neosynthesis of GABA was unique, dose dependent, and not tetrodotoxin sensitive. A total of 15 compounds (including standard, new and candidate anticonvulsants) either had no effect on new GABA or decreased it. Therefore, D-23129 and D-20443 exhibited two different effects on de novo synthesis of neurotransmitter amino acids, both of which could potentially be anticonvulsant in nature.

title: Pharmacogenetic Analysis Reveals a Post-Developmental Role for Rac GTPases in Caenorhabditis elegans GABAergic Neurotransmission
abstract: The nerve-cell cytoskeleton is essential for the regulation of intrinsic neuronal activity. For example, neuronal migration defects are associated with microtubule regulators, such as LIS1 and dynein, as well as with actin regulators, including Rac GTPases and integrins, and have been thought to underlie epileptic seizures in patients with cortical malformations. However, it is plausible that post-developmental functions of specific cytoskeletal regulators contribute to the more transient nature of aberrant neuronal activity and could be masked by developmental anomalies. Accordingly, our previous results have illuminated functional roles, distinct from developmental contributions, for Caenorhabditis elegans orthologs of LIS1 and dynein in GABAergic synaptic vesicle transport. Here, we report that C. elegans with function-altering mutations in canonical Rac GTPase-signaling-pathway members demonstrated a robust behavioral response to a GABAA receptor antagonist, pentylenetetrazole. Rac mutants also exhibited hypersensitivity to an acetylcholinesterase inhibitor, aldicarb, uncovering deficiencies in inhibitory neurotransmission. RNA interference targeting Rac hypomorphs revealed synergistic interactions between the dynein motor complex and some, but not all, members of Rac-signaling pathways. These genetic interactions are consistent with putative Rac-dependent regulation of actin and microtubule networks and suggest that some cytoskeletal regulators cooperate to uniquely govern neuronal synchrony through dynein-mediated GABAergic vesicle transport in C. elegans.

title: The ionic mechanism of the pentylenetetrazol convulsions.
abstract: The ionic dependence and the nature of conductance was examined at slowly inactivating inward current in metacerebral giant cells of Helix pomatia, induced by 50 mM pentylenetetrazol. Ramp and square wave depolarizations in voltage clamp mode revealed, that withdrawal of sodium ions prevented this current to flow. While TTX was ineffective, Mn, Co and Ni-ions and verapamil blocked the current. It is concluded that PTZ, especially in presence of TEA impairs calcium channels, which loose their specificity and transmit sodium ions, with very slow kinetics.

title: Seizure suppression by shakB2, a gap junction mutation in Drosophila.
abstract: Gap junction proteins mediate electrical synaptic transmission. In Drosophila, flies carrying null mutations in the shakB locus, such as shakB2, have behavioral and electrophysiological defects in the giant fiber (GF) system neurocircuit consistent with a loss of transmission at electrical synapses. The shakB2 mutation also affects seizure susceptibility. Mutant flies are especially seizure-resistant and have a high threshold to evoked seizures. In addition, in some double mutant combinations with "epilepsy" mutations, shakB2 appears to act as a seizure-suppressor mutation: shakB2 restores seizure susceptibility to the wild-type range in the double mutant. In double mutant combinations, shakB2 completely suppresses seizures caused by slamdance (sda), knockdown (kdn), and jitterbug (jbug) mutations. Seizures caused by easily shocked (eas) and technical knockout (tko) mutations are partially suppressed by shakB2. Seizures caused by bang-sensitive (bas2) and bang-senseless (bss1, bss2 alleles) mutations are not suppressed by shakB2. These results show the use of Drosophila as a model system for studying the kinds of genetic interactions responsible for seizure susceptibility, bringing us closer to unraveling the complexity of seizure disorders in humans.