Forebrain Electrophysiological Recording in Larval Zebrafish

Summary

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.

Abstract

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 system^1-3 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 zebrafish^5-7. 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.

Protocol

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 water. Unfertilized eggs and debris are removed using a transfer pipette.
3. Place Petri dish containing the collected eggs in an incubator (28-32 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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