Electrophysiological and pharmacological characterization of spreading depolarization in the adult zebrafish tectum

Spreading depolarization (SD) is a slowly propagating wave of neuronal and glial depolarization. A growing number of studies show that SD and SD-like phenomena play a role in neurological disorders such as migraine, stroke, and traumatic brain injury. Despite the clinical importance of SD, its underlying molecular and cellular mechanisms remain elusive, possibly because of insufficient animal model allowing genetic manipulation. Such a model would also allow high-throughput screening for SD-suppressing drug development. To address this, we developed a novel experimental system to study SD using zebrafish. Electrophysiological recordings in the immobilized adult zebrafish revealed that increasing extracellular potassium concentration elicited SD with a large and long-lasting negative shift of direct current (DC) potential in the optic tectum. It also reduced the oscillatory activity in the extracellular field potential and increased the expression of the immediate early gene c-fos. Pharmacological blocking of the N-methyl-d-aspartate (NMDA) glutamate receptor attenuated the propagation of SD, suggesting that glutamatergic neurotransmission mediated tectal SD in zebrafish. Our analyses revealed that the zebrafish tectum and rodent cortex had similar SD kinetics. The current study provides electrophysiological and pharmacological evidence that zebrafish SD and mammal SD are comparable. This zebrafish SD model is suitable for genetic manipulation and cost-effective high-throughput screening. It could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.

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Pericardial Injection of Kainic Acid Induces a Chronic Epileptic State in Larval Zebrafish

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.753936 ◽

2021 ◽

Vol 14 ◽

Author(s):

Lise Heylen ◽

Duc-Hung Pham ◽

Ann-Sofie De Meulemeester ◽

Éric Samarut ◽

Adrianna Skiba ◽

...

Keyword(s):

Kainic Acid ◽

High Throughput ◽

High Throughput Screening ◽

Video Recording ◽

Field Potential ◽

Latency Period ◽

Pharmacological Characterization ◽

Larval Zebrafish ◽

Zebrafish Larvae ◽

Human Pathology

Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which develop gradually during a process called epileptogenesis. The mechanistic processes underlying the changes of brain tissue and networks toward increased seizure susceptibility are not fully understood. In rodents, injection of kainic acid (KA) ultimately leads to the development of spontaneous epileptic seizures, reflecting similar neuropathological characteristics as seen in patients with temporal lobe epilepsy (TLE). Although this model has significantly contributed to increased knowledge of epileptogenesis, it is technically demanding, costly to operate and hence not suitable for high-throughput screening of anti-epileptic drugs (AEDs). Zebrafish, a vertebrate with complementary advantages to rodents, is an established animal model for epilepsy research. Here, we generated a novel KA-induced epilepsy model in zebrafish larvae that we functionally and pharmacologically validated. KA was administered by pericardial injection at an early zebrafish larval stage. The epileptic phenotype induced was examined by quantification of seizure-like behavior using automated video recording, and of epileptiform brain activity measured via local field potential (LFP) recordings. We also assessed GFP-labeled GABAergic and RFP-labeled glutamatergic neurons in double transgenic KA-injected zebrafish larvae, and examined the GABA and glutamate levels in the larval heads by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). Finally, KA-injected larvae were exposed to five commonly used AEDs by immersion for pharmacological characterization of the model. Shortly after injection, KA induced a massive damage and inflammation in the zebrafish brain and seizure-like locomotor behavior. An abnormal reorganization of brain circuits was observed, a decrease in both GABAergic and glutamatergic neuronal population and their associated neurotransmitters. Importantly, these changes were accompanied by spontaneous and continuous epileptiform brain discharges starting after a short latency period, as seen in KA rodent models and reminiscent of human pathology. Three out of five AEDs tested rescued LFP abnormalities but did not affect the seizure-like behavior. Taken together, for the first time we describe a chemically-induced larval zebrafish epilepsy model offering unique insights into studying epileptogenic processes in vivo and suitable for high-throughput AED screening purposes and rapid genetic investigations.

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In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00960.2005 ◽

2006 ◽

Vol 291 (1) ◽

pp. H269-H273 ◽

Cited By ~ 138

Author(s):

David J. Milan ◽

Ian L. Jones ◽

Patrick T. Ellinor ◽

Calum A. MacRae

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Larval Fish ◽

Low Noise ◽

Qt Prolongation ◽

Motion Artifacts ◽

Adult Zebrafish ◽

Perfusion System ◽

Drug Induced

In the last decade the zebrafish has become a major model organism for the study of development and organogenesis. To maximize the experimental utility of this organism, it will be important to establish methods for adult phenotyping. We previously proposed that the embryonic zebrafish may be useful in high-throughput screening for drug-induced cardiotoxicity. We now describe a method for the reproducible recording of the adult zebrafish ECG and illustrate its application in the investigation of QT-prolonging drugs. Zebrafish ECGs were obtained by inserting two needle electrodes through the ventral epidermis. Fish were perfused orally, and motion artifacts were eliminated with a paralytic dose of μ-conotoxin GIIIB. Test compounds were delivered via the perfusion system. Without a means of hydration and oxygenation, the fish succumb rapidly. The use of a perfusion system allowed stable recording for >6 h. Baseline conduction intervals were as follows: PR, 66 ms (SD 14); QRS, 34 ms (SD 11); QT, 242 ms (SD 54); and R-R, 398 ms (SD 77). The known QT-prolonging agents astemizole, haloperidol, pimozide, and terfenadine caused corrected QT increases of 18% (SD 9), 16% (SD 11), 17% (SD 9), and 11% (SD 6), respectively. The control drugs clonidine, penicillin and propranolol did not prolong the corrected QT interval. In conclusion, perfusion and muscular paralysis allows stable, low-noise recording of zebrafish ECGs. Agents known to cause QT prolongation in humans caused QT prolongation in fish in each case. The development of rigorous tools for the phenotyping of adult zebrafish will complement the high-throughput assays currently under development for embryonic and larval fish.

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Novel pre-clinical model for CDKL5 Deficiency Disorder

10.1101/2021.04.07.438335 ◽

2021 ◽

Author(s):

Rita J Serrano ◽

Clara Lee ◽

Robert J Bryson-Richardson ◽

Tamar Sztal

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Genetic Manipulation ◽

Muscle Tone ◽

Neurological Diseases ◽

Epileptic Seizures ◽

Loss Of Function ◽

Zebrafish Model ◽

Clinical Model

Cyclin-dependent kinase-like-5 (CDKL5) Deficiency Disorder (CDD) is a severe X-linked neurodegenerative disease characterized by early-onset epileptic seizures, low muscle tone, progressive intellectual disability, severe motor function and visual impairment. CDD affects approximately 1 in 60,000 live births with many patients dying by early adulthood. For many patients, quality of life is significantly reduced due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer a number of unique advantages for high-throughput pre-clinical evaluation of potential therapies for human neurological diseases including CDD. In particular, the large number of zebrafish that can be produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss of function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model to investigate CDD disease pathophysiology and allow high-throughput screening for effective therapies.

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Novel pre-clinical model for CDKL5 Deficiency Disorder

Disease Models & Mechanisms ◽

10.1242/dmm.049094 ◽

2021 ◽

Author(s):

Rita J. Serrano ◽

Clara Lee ◽

Alon M. Douek ◽

Jan Kaslin ◽

Robert J. Bryson-Richardson ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Genetic Manipulation ◽

Muscle Tone ◽

Neurological Diseases ◽

Epileptic Seizures ◽

Loss Of Function ◽

Zebrafish Model ◽

Clinical Model

Cyclin-Dependent Kinase-Like-5 (CDKL5) Deficiency Disorder (CDD) is a severe X-linked neurodegenerative disease characterized by early-onset epileptic seizures, low muscle tone, progressive intellectual disability, and severe motor function. CDD affects approximately 1 in 60,000 live births with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput pre-clinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss of function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model to investigate CDD disease pathophysiology and allow high-throughput screening for effective therapies.

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New High-Throughput Screening Assay To Reveal Similarities and Differences in Inhibitory Sensitivities of Multidrug ATP-Binding Cassette Transporters

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00956-08 ◽

2009 ◽

Vol 53 (4) ◽

pp. 1516-1527 ◽

Cited By ~ 16

Author(s):

Marcin Kolaczkowski ◽

Anna Kolaczkowska ◽

Noboru Motohashi ◽

Krystyna Michalak

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Plasma Membranes ◽

Dynamic Range ◽

Genetic Manipulation ◽

High Sensitivity ◽

Atp Binding ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Atp Binding Cassette

ABSTRACT Cdr1p is the major ATP-binding cassette multidrug transporter conferring resistance to azoles and other antifungals in Candida albicans. In this study, the identification of new Cdr1p inhibitors by use of a newly developed high-throughput fluorescence-based assay is reported. The assay also allowed monitoring of the activity and inhibition of the related transporters Pdr5p and Snq2p of Saccharomyces cerevisiae, which made it possible to compare its performance with those of previously established procedures. A high sensitivity, resulting from a wide dynamic range, was achieved upon high-level expression of the Cdr1p, Pdr5p, and Snq2p transporters in an S. cerevisiae strain in which the endogenous interfering activities were further reduced by genetic manipulation. An analysis of a set of therapeutically used and newly synthesized phenothiazine derivatives revealed different pharmacological profiles for Cdr1p, Pdr5p, and Snq2p. All transporters showed similar sensitivities to M961 inhibition. In contrast, Cdr1p was less sensitive to inhibition by fluphenazine, whereas phenothiazine selectively inhibited Snq2p. The inhibition potencies measured by the new assay reflected the ability of the compounds to potentiate the antifungal effect of ketoconazole (KTC), which was detoxified by the overproduced transporters. They also correlated with the 50% inhibitory concentration for inhibition of Pdr5p-mediated transport of rhodamine 6G in isolated plasma membranes. The most active derivative, M961, potentiated the activity of KTC against an azole-resistant CDR1-overexpressing C. albicans isolate.

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