Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Summary

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Abstract

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express¹ . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons^{2, 3} .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property^4-6, the frequency of which is modulated by visual cues from conspecifics² and the neuropeptide kisspeptin 1⁵. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein⁷ to show you how to identify neurons and monitor their electrical activity in the whole brain preparation⁶.

Protocol

1. Dissection of Brains from Adult Medaka

1. Anesthetize adult male or female (Figure 1A) by immersing in 5 ml MS-222 (150 mg/L, pH 7.4); wait a couple of minutes after gills movements have ceased before decapitating. All procedures were approved by the Institutional Animal Care and Use Committee of University of California-Los Angeles.
2. Decapitate the fish in fish saline at the caudal end of the operculum with scissors in a 60-mm diameter Petri dish.
3. Transfer fish head to a 35-mm diameter Petri dish, half filled with fish saline and lined with Sylgard that has a depression in it to position and secure the head for brain di....

Results

An example of bilateral clusters of GFP-labeled TN-GnRH3 neurons from the excised brain of medaka fish are shown in Figures 1B and 1C. Each cluster contains about 8-10 GnRH neurons. The spontaneous neuronal activities of the target TN-GnRH3 were recorded in current-clamp mode (I=0) with typical firing rates of 0.5-6 Hz. The pattern of action potential firing is typically a tonic or beating pattern, with a fairly regular interspike interval. Sample traces are shown in Figure 2

Discussion

GnRH3:GFP transgenic fishes provide unique models to study the neurophysiological mechanisms underlying neuronal integration and regulation in central control of behaviors that are both directly and indirectly involved in reproduction^{3, 8-10}. One of the significant advantages of this model system is that many GnRH3 neurons expressing GFP are close to the ventral surface of the brain, allowing for relatively easy access to the neurons for electrophysiological recording without disrupting neural circuit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microscope	Olympus		BX50W (Upright)
Amplifier	Axon Instruments		Axoclamp 200B
A-D converter	Computer Interference Corp.		Digidata ITC-18
Cooled CCD camera	PCO Computer Optics		Sensicam
Xenon lamp	Sutter Instruments Co.
GFP filter set	Chroma Technologies
Imaging Software	Intelligent Imaging Innovations		Slidebook software
Electrophysiology Data Acquisition Software	Axon Instruments		Axograph software
Electrophysiology Data Acquisition Software	AD Instruments Inc.		PowerLab
Headstage for electrophysiology	Axon Instruments		CV 203BU
Micromanipulator	Sutter Instrument Co	MP-285
Recording Chamber Platform	Warner Instrument Corp.		P1
Recording Chamber	Warner Instrument Corp.		RC-26G
Electrode Puller	Sutter instruments		P87
Filament for electrode puller	Sutter Instruments	FB330B	3.0 mm wide trough filament
1.5 mm glass capillaries	World Precision Instruments	1B150-4	Micr–lectrode for recording
Syringe	Becton Dickinson	309586	3 ml
MS-222	Sigma	E10521-10G	Ethyl 3-aminobenzoate methanesulfonate salt
Fish saline			mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES
Electrode solution (loose-patch)			mM: 150 NaCl; 3.5 KCl; 2.5 CaCl2; 1.3 MgCl2; 10 HEPES; 10 glucose
Electrode solution (whole-cell patch)			mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl2; 1 MgCl2; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine