JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Forebrain Electrophysiological Recording in Larval Zebrafish

Published: January 24th, 2013

DOI:

10.3791/50104

1Epilepsy Research Laboratory, Department of Neurological Surgery, University of California, San Francisco

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 μl during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

1. Egg Production and Collection

  1. Zebrafish husbandry follows standard procedures described previously8. Briefly, adult zebrafish are set up in breeding tanks with dividers in place. When the lights in the room come on the following morning, dividers are removed from breeding tanks and fish are allowed approximately 20 to 60 min of undisturbed mating time.
  2. Eggs from breeding tanks are collected in a strainer and rinsed with egg water. Eggs are then transferred to a Petri dish with egg wate.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Examples of electrographic seizure-like discharge recorded in the forebrain of an agar-embedded zebrafish larvae are shown in Figure 1. Large-amplitude multi-spike burst discharge in these samples was evoked by bath application of a convulsant drug, 40 mM pilocarpine (in A; 6 dpf) or 1 mM picrotoxin (in B; 8 dpf). In these recordings, immobilized and agar-embedded zebrafish are continuously monitored for up to 90 min. Fish remain viable under these recording conditions for up to 24 hr. Drugs are added to.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The extracellular recording method presented here enables a very sensitive and rapid analysis of brain activity. These recordings are analogous to electroencephalographic (EEG) monitoring commonly used to evaluate the presence of abnormal electrical discharge (i.e., seizure) in rodent models of epilepsy11 and patients12. Extracellular recordings can be combined with pharmacological manipulations, as shown here. These types of recordings can also be used to evaluate potential epileptic pheno.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The author would like to thank Peter Castro and Matthew Dinday for their early efforts to establish zebrafish in the laboratory. This work was funded by the National Institutes of Health EUREKA grant (#R01NS079214-01).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
Agarose low melting Fisher-Scientific BP1360-100 Dissolve in embryo media at 1.2%
Recording media Fisher-Scientific BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 1 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1.2 mM MgCl2, 10 mM Dextrose, 2.1 mM CaCl2
pH to approximately 7.3 with 1 N NaOH
Tricaine Argent Labs MS-222 0.02%
α-bungarotoxin Tocris Bioscience 2133 1 mg/ml
Capillary glass tubing Warner Instruments G120TF-3 Pull to a resistance of 2 -7 MΩ
Patch clamp amplifier Warner Instruments PC-505B We use a Warner amplifier in current-clamp mode; Gain set at 2 mV/pA and Bessel filter set at 2K. Comparable models can be used according to manufacturer's instructions.
Filter/amplifier Cygnus Technology FLA-01 We use a Cygnus pre-amplifier; Gain set at 10-20; Cut-off frequency set at 1-2K; Notch filter IN. Comparable models can be used according to manufacturer's instructions.
Axon A/D board and Axoscope software Molecular Devices Axon Digidata 1320A; Axoscope 8.2 Data is collected in Axoscope using gap-free acquisition mode; sampling at 10 kHz. Comparable models and programs can be used according to manufacturer's instructions.
Egg water Instant Ocean   3 g Instant Ocean sea salt, 2 ml 0.1% methylene blue in 10 ml deionized water

  1. Clark, K. J., et al. Stressing zebrafish for behavioral genetics. Reviews in Neuroscience. 22 (1), 49 (2011).
  2. Rinkwitz, S., et al. Zebrafish: an integrative system for neurogenomics and neurosciences. Progress in Neurobiology. 93 (2), 231 (2011).
  3. Penberthy, W. T., et al. The zebrafish as a model for human disease. Frontiers in Bioscience. 7, d1439 (2002).
  4. Letamendia, A., et al. Development and validation of an automated high-throughput system for zebrafish in vivo screenings. PLoS One. 7, e36690 (2012).
  5. Nasevicius, A., Ekker, S. C. Effective targeted gene 'knockdown' in zebrafish. Nature Genetics. 26 (2), 216 (2000).
  6. Haffter, P., et al. Mutations affecting development of the zebrafish inner ear and lateral line. Development. 123, 1 (1996).
  7. Suster, M. L., et al. Transgenesis in zebrafish with the tol2 transposon system. Methods Molecular Biology. 561, 41 (2009).
  8. Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of Zebrafish Embryos to Analyze Gene Function. J. Vis. Exp. (25), e1115 (2009).
  9. Baraban, S. C., et al. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience. 131 (3), 759 (2005).
  10. Baraban, S. C., et al. A large-scale mutagenesis screen to identify seizure-resistant zebrafish. Epilepsia. 48 (6), 1151 (2007).
  11. Williams, P., et al. The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy. Journal of Neuroscience Methods. 155 (1), 39 (2006).
  12. Marsh, E. D., et al. Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients. Epilepsia. 51 (4), 592 (2010).
  13. Zhu, C., et al. Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish. Development. 138 (20), 4555 (2011).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved