117 research outputs found
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Network Properties Revealed during Multi-Scale Calcium Imaging of Seizure Activity in Zebrafish.
Seizures are characterized by hypersynchronization of neuronal networks. Understanding these networks could provide a critical window for therapeutic control of recurrent seizure activity, i.e., epilepsy. However, imaging seizure networks has largely been limited to microcircuits in vitro or small "windows" in vivo. Here, we combine fast confocal imaging of genetically encoded calcium indicator (GCaMP)-expressing larval zebrafish with local field potential (LFP) recordings to study epileptiform events at whole-brain and single-neuron levels in vivo. Using an acute seizure model (pentylenetetrazole, PTZ), we reliably observed recurrent electrographic ictal-like events associated with generalized activation of all major brain regions and uncovered a well-preserved anterior-to-posterior seizure propagation pattern. We also examined brain-wide network synchronization and spatiotemporal patterns of neuronal activity in the optic tectum microcircuit. Brain-wide and single-neuronal level analysis of PTZ-exposed and 4-aminopyridine (4-AP)-exposed zebrafish revealed distinct network dynamics associated with seizure and non-seizure hyperexcitable states, respectively. Neuronal ensembles, comprised of coactive neurons, were also uncovered during interictal-like periods. Taken together, these results demonstrate that macro- and micro-network calcium motifs in zebrafish may provide a greater understanding of epilepsy
Medial Ganglionic Eminence Progenitors Transplanted into Hippocampus Integrate in a Functional and Subtype-Appropriate Manner.
Medial ganglionic eminence (MGE) transplantation rescues disease phenotypes in various preclinical models with interneuron deficiency or dysfunction, including epilepsy. While underlying mechanism(s) remains unclear to date, a simple explanation is that appropriate synaptic integration of MGE-derived interneurons elevates GABA-mediated inhibition and modifies the firing activity of excitatory neurons in the host brain. However, given the complexity of interneurons and potential for transplant-derived interneurons to integrate or alter the host network in unexpected ways, it remains unexplored whether synaptic connections formed by transplant-derived interneurons safely mirror those associated with endogenous interneurons. Here, we combined optogenetics, interneuron-specific Cre driver mouse lines, and electrophysiology to study synaptic integration of MGE progenitors. We demonstrated that MGE-derived interneurons, when transplanted into the hippocampus of neonatal mice, migrate in the host brain, differentiate to mature inhibitory interneurons, and form appropriate synaptic connections with native pyramidal neurons. Endogenous and transplant-derived MGE progenitors preferentially formed inhibitory synaptic connections onto pyramidal neurons but not endogenous interneurons. These findings demonstrate that transplanted MGE progenitors functionally integrate into the postnatal hippocampal network
Behavioral Comorbidities and Drug Treatments in a Zebrafish scn1lab Model of Dravet Syndrome.
Loss-of-function mutations in SCN1A cause Dravet syndrome (DS), a catastrophic childhood epilepsy in which patients experience comorbid behavioral conditions, including movement disorders, sleep abnormalities, anxiety, and intellectual disability. To study the functional consequences of voltage-gated sodium channel mutations, we use zebrafish with a loss-of-function mutation in scn1lab, a zebrafish homolog of human SCN1A. Homozygous scn1labs552/s552 mutants exhibit early-life seizures, metabolic deficits, and early death. Here, we developed in vivo assays using scn1labs552 mutants between 3 and 6 d postfertilization (dpf). To evaluate sleep disturbances, we monitored larvae for 24 h with locomotion tracking software. Locomotor activity during dark (night phase) was significantly higher in mutants than in controls. Among anticonvulsant drugs, clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved at night for scn1labs552 mutant larvae. To monitor exploratory behavior in an open field, we tracked larvae in a novel arena. Mutant larvae exhibited impaired exploratory behavior, with increased time spent near the edge of the arena and decreased mobility, suggesting greater anxiety. Both clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved and increased time spent in the center of the arena. Counting inhibitory neurons in vivo revealed no differences between scn1labs552 mutants and siblings. Taken together, our results demonstrate conserved features of sleep, anxiety, and movement disorders in scn1lab mutant zebrafish, and provide evidence that a zebrafish model allows effective tests of treatments for behavioral comorbidities associated with DS
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Phenotype-Based Screening of Synthetic Cannabinoids in a Dravet Syndrome Zebrafish Model.
Dravet syndrome is a catastrophic epilepsy of childhood, characterized by cognitive impairment, severe seizures, and increased risk for sudden unexplained death in epilepsy (SUDEP). Although refractory to conventional antiepileptic drugs, emerging preclinical and clinical evidence suggests that modulation of the endocannabinoid system could be therapeutic in these patients. Preclinical research on this topic is limited as cannabis, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), are designated by United States Drug Enforcement Agency (DEA) as illegal substances. In this study, we used a validated zebrafish model of Dravet syndrome, scn1lab homozygous mutants, to screen for anti-seizure activity in a commercially available library containing 370 synthetic cannabinoid (SC) compounds. SCs are intended for experimental use and not restricted by DEA designations. Primary phenotype-based screening was performed using a locomotion-based assay in 96-well plates, and a secondary local field potential recording assay was then used to confirm suppression of electrographic epileptiform events. Identified SCs with anti-seizure activity, in both assays, included five SCs structurally classified as indole-based cannabinoids JWH 018 N-(5-chloropentyl) analog, JWH 018 N-(2-methylbutyl) isomer, 5-fluoro PB-22 5-hydroxyisoquinoline isomer, 5-fluoro ADBICA, and AB-FUBINACA 3-fluorobenzyl isomer. Our approach demonstrates that two-stage phenotype-based screening in a zebrafish model of Dravet syndrome successfully identifies SCs with anti-seizure activity
A Novel Long-term, Multi-Channel and Non-invasive Electrophysiology Platform for Zebrafish.
Zebrafish are a popular vertebrate model for human neurological disorders and drug discovery. Although fecundity, breeding convenience, genetic homology and optical transparency have been key advantages, laborious and invasive procedures are required for electrophysiological studies. Using an electrode-integrated microfluidic system, here we demonstrate a novel multichannel electrophysiology unit to record multiple zebrafish. This platform allows spontaneous alignment of zebrafish and maintains, over days, close contact between head and multiple surface electrodes, enabling non-invasive long-term electroencephalographic recording. First, we demonstrate that electrographic seizure events, induced by pentylenetetrazole, can be reliably distinguished from eye or tail movement artifacts, and quantifiably identified with our unique algorithm. Second, we show long-term monitoring during epileptogenic progression in a scn1lab mutant recapitulating human Dravet syndrome. Third, we provide an example of cross-over pharmacology antiepileptic drug testing. Such promising features of this integrated microfluidic platform will greatly facilitate high-throughput drug screening and electrophysiological characterization of epileptic zebrafish
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Zebrafish studies identify serotonin receptors mediating antiepileptic activity in Dravet syndrome.
Dravet syndrome is a life-threatening early-onset epilepsy not well controlled by antiepileptic drugs. Drugs that modulate serotonin (5-HT) signalling, including clemizole, locaserin, trazodone and fenfluramine, have recently emerged as potential treatment options for Dravet syndrome. To investigate the serotonin receptors that could moderate this antiepileptic activity, we designed and synthesized 28 novel analogues of clemizole, obtained receptor binding affinity profiles, and performed in vivo screening in a scn1lab mutant zebrafish (Danio rerio) model which recapitulates critical clinical features of Dravet syndrome. We discovered three clemizole analogues with 5-HT receptor binding that exert powerful antiepileptic activity. Based on structure-activity relationships and medicinal chemistry-based analysis, we then screened an additional set of known 5-HT receptor specific drug candidates. Integrating our in vitro and in vivo data implicates 5-HT2B receptors as a critical mediator in the mechanism of seizure suppression observed in Dravet syndrome patients treated with 5-HT modulating drugs
Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.
Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations
Impaired neural development in a zebrafish model for Lowe syndrome
Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome
GABAB receptors in maintenance of neocortical circuit function
Activation of metabotropic GABA(B) receptors (GABA(B)Rs), which enhances tonic GABA current, substantially increases the frequency of spontaneous seizures. Despite these pro-epileptic consequences of GABA(B)R activation, mice lacking functional GABA(B) receptors (GABA(B1)R KO mice) exhibit clonic and rare absence seizures. To examine these mice further, we recorded excitatory and inhibitory synaptic inputs and tonic GABA currents from Layer 2 neocortical pyramidal neurons of GABA(B1)R WT and KO mice (P30-40). Tonic current was increased while the frequency of synaptic inputs was unchanged in KO mice relative to WT littermates. The neocortical laminar distribution of interneuron subtypes derived from the medial ganglionic eminence (MGE) was also not statistically different in KO mice relative to WT while the number of calretinin-positive, caudal GEderived, cells in Layer 1 was reduced. Transplantation of MGE progenitors obtained from KO mice lacking functional GABA(B1)R did not increase tonic inhibition in the host brain above that of media-injected controls. Taken together, these results suggest a complex role for GABA(B) receptors in mediating neocortical circuit function
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