In Vivo Whole-cell Patch-clamp Recording of Hypothalamic Peptidergic Neurons in Larval Zebrafish

Summary

This article presents a protocol for in vivo recording of electrical activity of hypothalamic peptidergic neurons using whole-cell patch-clamp electrophysiology in intact larval zebrafish.

Abstract

The hypothalamus is an ancient brain region that regulates diverse aspects of physiology and behavior, including sleep and wakefulness, appetite, energy homeostasis, anxiety, depression, and social interaction. Specific neuronal populations in the hypothalamus exert their effects via the release of neurotransmitters and neuropeptides. Whole-cell patch-clamp recording is an indispensable approach for studying the roles of these factors in synaptic transmission and brain function. However, it is challenging to access hypothalamic neurons for electrophysiological recordings in intact mammals due to their location deep within the brain. As a result, our understanding of the intrinsic properties and physiological functions of hypothalamic neurons is limited. The larval zebrafish is a useful alternative model to study hypothalamic neurons due to its transparent and small, but well-conserved, vertebrate brain. Here, we present a protocol for in vivo whole-cell patch clamp recordings of hypothalamic neurons in intact larval zebrafish. Using this technique, we can record from peptidergic neurons in the hypothalamus, examine the responses of these neurons to sensory stimuli, and explore their effects on downstream neurons. This experimental technique thus provides a useful approach to study the physiological functions of hypothalamic neuropeptidergic neurons in intact animals.

Introduction

As a popular vertebrate animal model, zebrafish (Danio rerio) are widely used in neuroscience research and have several advantages¹. Their complement of genes and brain architecture is highly conserved with mammals, and they have a rich behavioral repertoire, making them useful to study genetic and neuronal mechanisms that underlie behaviors such as sleep, anxiety, depression, and social interaction^2,3,4,5,6. Their small size and low maintenance costs make them ideal for hig....

Protocol

All experiments were approved by the Institutional Care and Use Committee (IACUC) at Shanghai University (animal protocol YS 2025-169) and IACUC at California Institute of Technology (animal protocol 1836). Zebrafish from 5 to 7 days post-fertilization (dpf) were fed with rotifers and used for experiments. At this stage of development, sex is not determined. Adult zebrafish on a nacre [mitfa(w2/w2)] background²⁴ were used for breeding.

1. Solutions and recipes

1. Prepare E3 medium containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄. Adjust the solution b....

Results

In this manuscript, we present an improved in vivo whole-cell patch-clamp recording technique for investigating hypothalamic neurons in intact zebrafish, particularly focusing on hypocretin (Hcrt) neurons.

As presented in Figure 1, our methodology enables direct electrophysiological recordings from these neurons deep within the brain in an intact animal (Figure 1A), overcoming limitations of traditional in vitro slic.......

Discussion

The protocol described here enables patch-clamp recordings of peptidergic neurons in the larval zebrafish hypothalamus, one of the deepest and most technically challenging brain regions to access. Due to the inherent difficulty of this preparation, successful patch-clamp recordings require meticulous attention to a few critical parameters, namely pipette quality, approach technique, solution purity, and tissue health. These factors collectively determine the likelihood of achieving and maintaining stable giga-seal record.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amplifier	Axon	700B
Borosilicate glass capillaries	Sutter	BF100-58-10
CCD camera	Dage-MTI	IR-1000
Computers for electrophysiological recordings	Dell	Precision 3660
Digidata	Axon	1440A
Faraday Cage	Custom-made
Forceps	F.S.T.	Dumont #5
Incubator	Lonroy	GZP-150B
Membrane filter	Millipore Sigma	SLGV004SL
Micro knife	F.S.T.	10318-14
Objective	Olympus	Mplan 5X/0.1; UMPlanFI/IR 60X/0.9w
Peristaltic pump	Longer	BT100-1L
Pipette holder	Narishige	H-7	for dissection
Puller	Sutter Instrument	p-97
Stereomicroscope for fluorescent screening and dissection	Olympus	SZX16
Stimulator	A.M.P.I	Master8
Three-dimensional micromanipulator	Sutter Instrument	MPC-325
Upright infrared DIC microscope	Olympus	BX51WI
Vibration isolator table	TMC	61-541-06
Video monitor	SUNSPO	SP-717
Water bath	Yiheng	HWS-12
X-Y translator	Custom-made