Transcutaneous Vagus Nerve Stimulation (tVNS) for Treatment of Drug-Resistant Epilepsy: A Randomized, Double-Blind Clinical Trial (cMPsE02).

BACKGROUND: Various brain stimulation techniques are in use to treat epilepsy. These methods usually require surgical implantation procedures. Transcutaneous vagus nerve stimulation (tVNS) is a non-invasive technique to stimulate the left auricular branch of the vagus nerve at the ear conch. OBJECTIVE: We performed a randomized, double-blind controlled trial (cMPsE02) to assess efficacy and safety of tVNS vs. control stimulation in patients with drug-resistant epilepsy. METHODS: Primary objective was to demonstrate superiority of add-on therapy with tVNS (stimulation frequency 25 Hz, n = 39) versus active control (1 Hz, n = 37) in reducing seizure frequency over 20 weeks. Secondary objectives comprised reduction in seizure frequency from baseline to end of treatment, subgroup analyses and safety evaluation. RESULTS: Treatment adherence was 84% in the 1 Hz group and 88% in the 25 Hz group, respectively. Stimulation intensity significantly differed between the 1 Hz group (1.02 ± 0.83 mA) and the 25 Hz group (0.50 ± 0.47 mA; p = 0.006). Mean seizure reduction per 28 days at end of treatment was -2.9% in the 1 Hz group and 23.4% in the 25 Hz group (p = 0.146). In contrast to controls, we found a significant reduction in seizure frequency in patients of the 25 Hz group who completed the full treatment period (20 weeks; n = 26, 34.2%, p = 0.034). Responder rates (25%, 50%) were similar in both groups. Subgroup analyses for seizure type and baseline seizure frequency revealed no significant differences. Adverse events were usually mild or moderate and comprised headache, ear pain, application site erythema, vertigo, fatigue, and nausea. Four serious adverse events were reported including one sudden unexplained death in epilepsy patients (SUDEP) in the 1 Hz group which was assessed as not treatment-related. CONCLUSIONS: tVNS had a high treatment adherence and was well tolerated. Superiority of 25 Hz tVNS over 1 Hz tVNS could not be proven in this relatively small study, which might be attributed to the higher stimulation intensity in the control group. Efficacy data revealed results that justify further trials with larger patient numbers and longer observation periods.

An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants.

[Epileptic seizures during treatment with antidepressants and neuroleptics]. / Epileptische Anfälle unter der Behandlung mit Antidepressiva und Neuroleptika.

Epileptic seizures are observed during treatment with antidepressants and neuroleptics more frequently than is the case for other neuroactive substances. Evidence from experimental and observational studies is mixed, suggesting an increased incidence of seizures for certain drugs, whilst other drugs such as SSRIs appear to have a protective effect. There is robust evidence for an elevated seizure incidence (up to 4.5 % of treated patients) associated with clozapine treatment, but with other neuroleptics the effect is moderate (2-fold). The evaluation of data from FDA approval reports reveals lower standardised incidence rates associated with antidepressants vs. placebo except for clomipramine and bupropione. Psychiatric disorders such as schizophrenia, depression, and obsessive-compulsive disorder are associated with a considerably increased incidence of seizures. Therefore, in clinical practice, taking into account ictogenic properties of substances is required only in patients with a history of seizures.

Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.

Rodent seizure models have significantly contributed to our basic understanding of epilepsy. However, medically intractable forms of epilepsy persist and the fundamental mechanisms underlying this disease remain unclear. Here we show that seizures can be elicited in a simple vertebrate system e.g. zebrafish larvae (Danio rerio). Exposure to a common convulsant agent (pentylenetetrazole, PTZ) induced a stereotyped and concentration-dependent sequence of behavioral changes culminating in clonus-like convulsions. Extracellular recordings from fish optic tectum revealed ictal and interictal-like electrographic discharges after application of PTZ, which could be blocked by tetrodotoxin or glutamate receptor antagonists. Epileptiform discharges were suppressed by commonly used antiepileptic drugs, valproate and diazepam, in a concentration-dependent manner. Up-regulation of c-fos expression was also observed in CNS structures of zebrafish exposed to PTZ. Taken together, these results demonstrate that chemically-induced seizures in zebrafish exhibit behavioral, electrographic, and molecular changes that would be expected from a rodent seizure model. Therefore, zebrafish larvae represent a powerful new system to study the underlying basis of seizure generation, epilepsy and epileptogenesis.

Generalized epilepsy with febrile seizures plus: further heterogeneity in a large family.

BACKGROUND: Generalized epilepsy with febrile seizures plus (GEFS(+)) is a recently described benign childhood-onset epileptic syndrome with autosomal dominant inheritance. The most common phenotypes are febrile seizures (FS) often with accessory afebrile generalized tonic-clonic seizures (GTCS, FS(+)). In about one third, additional seizure types occur, such as absences, myoclonic, or atonic seizures. So far, three mutations within genes encoding subunits of neuronal voltage-gated Na(+) channels have been found in GEFS(+) families, one in SCN1B (beta(1)-subunit) and two in SCN1A (alpha-subunit). METHODS: The authors examined the phenotypic variability of GEFS(+) in a five-generation German family with 18 affected individuals. Genetic linkage analysis was performed to exclude candidate loci. RESULTS: Inheritance was autosomal dominant with a penetrance of about 80%. A variety of epilepsy phenotypes occurred predominantly during childhood. Only four individuals showed the FS or FS(+) phenotype. The others presented with different combinations of GTCS, tonic seizures, atonic seizures, and absences, only in part associated with fever. The age at onset was 2.8 +/- 1.3 years. Interictal EEG recordings showed rare, 1- to 2-second-long generalized, irregular spike-and-wave discharges of 2.5 to 5 Hz in eight cases and additional focal parietal discharges in one case. Linkage analysis excluded the previously described loci on chromosomes 2q21-33 and 19q13. All other chromosomal regions containing known genes encoding neuronal Na(+) channel subunits on chromosomes 3p21-24, 11q23, and 12q13 and described loci for febrile convulsions on chromosomes 5q14-15, 8q13-21, and 19p13.3 were also excluded. CONCLUSION: These results indicate further clinical and genetic heterogeneity in GEFS(+).

Retinal ganglion cell genesis requires lakritz, a Zebrafish atonal Homolog.

Mutation of the zebrafish lakritz (lak) locus completely eliminates the earliest-born retinal cells, the ganglion cells (RGCs). Instead, excess amacrine, bipolar, and Müller glial cells are generated in the mutant. The extra amacrines are found at ectopic locations in the ganglion cell layer. Cone photoreceptors appear unaffected by the mutation. Molecular analysis reveals that lak encodes Ath5, the zebrafish eye-specific ortholog of the Drosophila basic helix-loop-helix transcription factor Atonal. A combined birth-dating and cell marker analysis demonstrates that lak/ath5 is essential for RGC determination during the first wave of neurogenesis in the retina. Our results suggest that this wave is skipped in the mutant, leading to an accumulation of progenitors for inner nuclear layer cells.

Perception of Fourier and non-Fourier motion by larval zebrafish.

A moving grating elicits innate optomotor behavior in zebrafish larvae; they swim in the direction of perceived motion. We took advantage of this behavior, using computer-animated displays, to determine what attributes of motion are extracted by the fish visual system. As in humans, first-order (luminance-defined or Fourier) signals dominated motion perception in fish; edges or other features had little or no effect when presented with these signals. Humans can see complex movements that lack first-order cues, an ability that is usually ascribed to higher-level processing in the visual cortex. Here we show that second-order (non-Fourier) motion displays induced optomotor behavior in zebrafish larvae, which do not have a cortex. We suggest that second-order motion is extracted early in the lower vertebrate visual pathway.

Zebrafish on the move: towards a behavior-genetic analysis of vertebrate vision.

Behavioral screens have uncovered dozens of zebrafish mutants with striking visual defects. In parallel with mutant studies, recent psychophysical experiments indicate that zebrafish are capable of high-level motion processing, previously thought to be restricted to animals with a visual cortex. It should be possible now to devise assays to screen for mutations in visual perception.

Combining physiology and genetics in the zebrafish retina.

The zebrafish has recently joined the ranks of Drosophila and C. elegans as a tractable model for genetic screens (Fishman, 1999). Zebrafish grow fast, can be kept in large numbers in a small space, and are efficiently mutagenized and screened. Genomic resources are made available at an increasing pace. These days, a mutation can be mapped and cloned in a matter of months. Because a mutant hunt is intrinsically unbiased in terms of the classes of genes that will be tagged, it holds the unique potential to discover novel genes or, in our era of genome sequencing, to identify novel functions for known genes. Zebrafish display dozens of innate behaviours in response to light, of which the optomotor and the optokinetic responses are the most widely studied (Brockerhoff et al. 1995; Easter & Nicola, 1996). Their retinae are crisply layered following the typical vertebrate pattern, and the retinal layers are tiled in an almost crystalline fashion by mosaics of different cell types. Electroretinograms are recorded routinely and therefore, not surprisingly, zebrafish are now also being used for a genetic approach to the visual system.

FTDP-17: an early-onset phenotype with parkinsonism and epileptic seizures caused by a novel mutation.

Recently, mutations in the tau gene on chromosome 17 were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism (FTDP-17). We describe a family carrying a missense mutation at nucleotide 1137 C --> T, resulting in the amino acid substitution P301S. Methods of investigations include clinical, electrophysiological, and imaging techniques. This kindred presents with a novel phenotype characterized by an early onset of rapidly progressive frontotemporal dementia and parkinsonism in combination with epileptic seizures. We define the dopaminergic deficits as being predominantly presynaptic by the use of single-photon emission computed tomography with a dopamine transporter ligand. The association of this early-onset phenotype with P301S mutation is not entirely consistent with current criteria for the diagnosis of frontotemporal dementias and may encourage the search for tau mutations in diseases similar but not identical to FTDP-17. Also, the change from proline to serine suggests that this mutation might contribute to tau hyperphosphorylation.

Genetic disorders of vision revealed by a behavioral screen of 400 essential loci in zebrafish.

We examined optokinetic and optomotor responses of 450 zebrafish mutants, which were isolated previously based on defects in organ formation, tissue patterning, pigmentation, axon guidance, or other visible phenotypes. These strains carry single point mutations in >400 essential loci. We asked which fraction of the mutants develop blindness or other types of impairments specific to the visual system. Twelve mutants failed to respond in either one or both of our assays. Subsequent histological and electroretinographic analysis revealed unique deficits at various stages of the visual pathway, including lens degeneration (bumper), melanin deficiency (sandy), lack of ganglion cells (lakritz), ipsilateral misrouting of axons (belladonna), optic-nerve disorganization (grumpy and sleepy), inner nuclear layer or outer plexiform layer malfunction (noir, dropje, and possibly steifftier), and disruption of retinotectal impulse activity (macho and blumenkohl). Surprisingly, mutants with abnormally large or small eyes or severe wiring defects frequently exhibit no discernible behavioral deficits. In addition, we identified 13 blind mutants that display outer-retina dystrophy, making this syndrome the single-most common cause of inherited blindness in zebrafish. Our screen showed that a significant fraction (approximately 5%) of the essential loci also participate in visual functions but did not reveal any systematic genetic linkage to particular morphological traits. The mutations uncovered by our behavioral assays provide distinct entry points for the study of visual pathways and set the stage for a genetic dissection of vertebrate vision.

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Axon guidance: stretching gradients to the limit.

Neuronal growth cones, the sensory-motile structures at the tips of developing axons, navigate to their targets over distances that can be many times greater than their diameter. They may accomplish this impressive task by following spatial gradients of axon guidance molecules in their environment (Bonhoeffer & Gierer, 1984; Tessier-Lavigne & Placzek, 1991; Baier & Bonhoeffer, 1994). We calculate the optimal shape of a gradient and the distance over which it can be detected by a growth cone for two competing mechanistic models of axon guidance. The results are surprisingly simple: Regardless of the mechanism, the maximum distance is about 1 cm. Since gradients and growth cones have coevolved, we suggest that the shape of the gradient in situ will predict the mechanism of gradient detection. In addition, we show that the experimentally determined dissociation constants for receptor-ligand complexes implicated in axon guidance are about optimal with respect to maximizing guidance distance. The relevance of these results to the retinotectal system is discussed.

Synchronization of neuronal activity promotes survival of individual rat neocortical neurons in early development.

Neural activity is thought to play a significant role during the development of the cerebral cortex. In this study, we examined the effects of global activity block or enhancement and the effects of patterned firing on the ability of cultured rat neocortical neurons to survive during the second week in vitro, beyond the beginning of synaptogenesis. Blockade of neuronal activity by adding tetrodotoxin (TTX) and increasing magnesium concentration in the medium strongly reduced the survival of cortical cells. Increasing neuronal activity by raising the external potassium concentration significantly improved the survival of cortical neurons. We postulated that in a developing neuronal network the survival of nerve cells is regulated by synaptically mediated events that involve changes in the intracellular calcium concentration. To examine this question further, we monitored the activity of the developing network by optically recording the intracellular calcium signals of many neurons simultaneously. These recordings show that in low magnesium neocortical neurons express synchronized oscillation of their intracellular calcium concentration. The ability of a network to synchronize the changes in intracellular calcium of multiple cells appeared gradually during the second week in culture, paralleled by both an increase in the synaptic density and a decline in the number of surviving neurons. By examining the fate of identified cells several days after a recording session, we found that those nerve cells that were co-activated with other neurons had a significantly higher chance to survive than cells that did not participate in synchronized events. These experiments demonstrate that during early cortical network development cortical neurons show synchronized firing activity and that the survival of neurons is at least partially dependent on this pattern of neuronal activity.

Genetic dissection of the retinotectal projection.

A systematic search for mutations affecting the retinotectal projection in zebrafish larvae was performed, as part of the large-scale Tubingen screen for homozygous diploid mutants in embryonic development. 2,746 inbred lines (F2 families) from males mutagenized with ethylnitroso urea were screened. In wild-type larvae, developing retinal axons travel along a stereotyped route to the contralateral optic tectum. Here, their terminals form a highly ordered retinotopic map. To detect deviations from this pattern, an axon tracing assay was developed that permits screening of large numbers of mutagenized fish. Two fluorescent tracer dyes (DiI and DiO) were injected at opposite poles of the eyes of day-5 aldehyde-fixed larvae. 12 hours later, retinal axons were labelled over their entire length, and could be observed through the intact skin. The assay procedure (aldehyde fixation, mounting, injection of dyes, microscopic analysis) took about 1 minute per fish. In total, 125,000 individual fish larvae were processed. During the screen, 114 mutations in approx. 35 genes were discovered. For the mutants subjected to complementation testing, the number of alleles per locus ranges from 1 to 15. The mutations affect distinct steps in the retinotectal pathway, from pathfinding between eye and tectum to map formation along the dorsal-ventral and the anterior-posterior axis of the tectum. Mutations that disturb axon pathfinding to the tectum for the most part do not disrupt retinotopic mapping, and vice versa. The majority of the mutants display associated defects in other tissues and die before day 10. These mutants provide new tools for studying the formation of neuronal maps. The results of this screen show that a large-scale genetic approach can be applied to relatively late and circumscribed developmental processes in the vertebrate brain.

Zebrafish mutations affecting retinotectal axon pathfinding.

We have isolated mutants in the zebrafish Danio rerio that have defects in axonal connectivity between the retina and tectum. 5-day-old fish larvae were screened by labeling retinal ganglion cells with DiI and DiO and observing their axonal projections to and on the tectum. 82 mutations, representing 13 complementation groups and 6 single allele loci, were found that have defects in retinal ganglion cell axon pathfinding to the tectum. These pathfinding genes fall into five classes, based on the location of pathfinding errors between eye and tectum. In Class I mutant larvae (belladonna, detour, you-too, iguana, umleitung, blowout) axons grow directly to the ipsilateral tectal lobe after leaving the eye. Class II mutant larvae (chameleon, bashful) have ipsilaterally projecting axons and, in addition, pathfinding mistakes are seen within the eye. In Class III mutant larvae (esrom, tilsit, tofu) fewer axons than normal cross the midline, but some axons do reach the contralateral tectal lobe. Class IV mutant larvae (boxer, dackel, pinscher) have defects in axon sorting after the midline and retinal axons occasionally make further pathfinding errors upon reaching the contralateral tectal lobe. Finally, Class V mutant larvae (bashful, grumpy, sleepy, cyclops, astray) have anterior-posterior axon trajectory defects at or after the midline. The analysis of these mutants supports several conclusions about the mechanisms of retinal axon pathfinding from eye to tectum. A series of sequential cues seems to guide retinal axons to the contralateral tectal lobe. Pre-existing axon tracts seem not to be necessary to guide axons across the midline. The midline itself seems to play a central role in guiding retinal axons. Axons in nearby regions of the brain seem to use different cues to cross the ventral midline. Mutant effects are not all-or-none, as misrouted axons may reach their target, and if they do, they project normally on the tectum. The retinotectal pathfinding mutants reveal important choice points encountered by neuronal growth cones as they navigate between eye and tectum.

Mutations disrupting the ordering and topographic mapping of axons in the retinotectal projection of the zebrafish, Danio rerio.

Retinal ganglion cells connect to their target organ, the rectum, in a highly ordered fashion. We performed a large-scale screen for mutations affecting the retinotectal projection of the zebrafish, which resulted in the identification of 114 mutations. 44 of these mutations disturb either the order of RGC axons in the optic nerve and tract, the establishment of a topographic map on the tectum, or the formation of proper termination fields. Mutations in three genes, boxer, dackel and pinscher, disrupt the sorting of axons in the optic tract but do not affect mapping on the tectum. In these mutants, axons from the dorsal retina grow along both the ventral and the dorsal branch of the optic tract. Mutations in two genes, nevermind and who-cares, affect the dorsoventral patterning of the projection. In embryos homozygous for either of these mutations, axons from dorsal retinal ganglion cells terminate ventrally and dorsally in the tectum. In nevermind, the retinotopic order of axons along the optic nerve and tract is changed in a characteristic way as well, while it appears to be unaffected in who-cares. Two mutations in two complementation groups, gnarled and macho, affect the anteroposterior patterning of the projection. In these mutants, nasodorsal axons branch and terminate too soon in the anterior tectum. In 27 mutants belonging to six complementation groups, retinal axons do not form normal termination fields. Some implications for models concerning the formation of topographic projections are discussed.