Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Summary

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

Abstract

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) in response to an action potential (AP). Since SVs are recycled locally at the presynaptic terminal, coordination of SV exocytosis and endocytosis is important for sustained synaptic transmission. A pH-sensitive green fluorescent protein, called pHluorin, provides a powerful tool to monitor SV exo/endocytosis by targeting it to the SV lumen. However, tracking AP-driven SV recycling with the pHluorin-based probes is still largely limited to in vitro culture preparations because the introduction of genetically encoded probes and subsequent optical imaging is technically challenging in general for in vivo animal models or tissue preparations. Zebrafish is a model system offering valuable features, including ease of genetic manipulation, optical clarity, and rapid external development. We recently generated a transgenic zebrafish that highly expresses a pHluorin-labeled probe at motor neuron terminals and developed a protocol to monitor AP-driven SV exo/endocytosis at the neuromuscular junction (NMJ), a well-established synapse model that forms in vivo. In this article, we show how to prepare larval zebrafish NMJ preparation suitable for pHluorin imaging. We also show that the preparation allows time-lapse imaging under conventional upright epifluorescence microscope, providing a cost-effective platform for analyzing NMJ function.

Introduction

Synaptic transmission, mediated by neurotransmitter release from synaptic vesicles (SVs) at the presynaptic terminal, is a fundamental process underlying nerve function¹. In early studies, synaptic transmission was measured primarily by electrophysiological techniques that detect the postsynaptic response elicited by neurotransmitters and their receptors. Over the past few decades, however, several types of imaging techniques have been developed that directly visualize presynaptic function². One of the most widely used probes is a pH-sensitive green fluorescent protein called pHluorin^3,....

Protocol

All animal procedures were conducted in accordance with the guidelines for the care and use of animals at Osaka Medical and Pharmaceutical University. Zebrafish were raised and maintained under a 14 h light to 10 h dark cycle. The embryos and larvae were maintained at 28-30 °C in egg water containing 0.006% sea salt and 0.01% methylene blue. The experiments were conducted at 4-7 days post-fertilization (dpf). It is recommended that the fish be fed twice a day from 5 dpf onwards when experiments are performed after 6 dpf. The medium must be changed prior to each feeding.

1. Preparation of solutions

1. Prepare 2....

Discussion

The larval zebrafish NMJ is an emerging model system for the study of synaptic physiology and pathology^26,31. A transgenic zebrafish expressing SpH in a neuron-specific manner has already been generated and employed for the analysis of a mutant exhibiting a locomotor defect¹⁷. Wen et al.¹⁷ demonstrated an approximately 2-fold increase in pHluorin fluorescence during stimulation of 1000 APs at 100 Hz in WT control NM.......

Materials

Name	Company	Catalog Number	Comments
40x water immersion objective	Olympus	LUMPLFLN40XW
4ch gravity flow perfusion system	ALA	VCPlus-4G
5x objective	Olympus	NPLN5X
Custum made imaging chamber	Physiotech	custum made	A black acrylic plate (10.7 cm diameter, 3 mm thick) with a well (1 cm diameter at the bottom, 1.5 cm diameter at the top) holding approximately 0.5 ml of perfusate.
Digital I/O device	Arduino	Uno Rev3
D-Tubocurarine dichloride pentahydrate	Sigma	93750
Excel	Microsoft	Microsoft Office Professional Plus 2016
Fiji / imageJ	https://imagej.net/	imageJ 1.54f
Fine forceps	AsOne	7-562-05 (Dumont #5)
Glass Pasteur pipette	IWAKI	IK-PAS-5P	The tip should be trimmed and fire-polished until the final diameter is 1.5–2 mm.
Glass Petri dish	AsOne	1-4564-06
Igor Pro	WaveMetrics	Ver. 6.37
Inline solution heater	Warner Instruments	SF-28
LED illumination system	X-cyte	XYLIS
Methylen blue	Wako	133-06962
Micro-Manager	https://micro-manager.org/	Ver. 2.0.0
Mini magnetic clamp for perfusion tube	Warner Instruments	64-1553
Motorized micromanipulator	Scientifica	PatchStar
Motorized movable sample plate	Scientifica	MMSP
Pipette holder	Narishige	H-13
Pipette puller	Narishige	PC-100
Platinum wire (φ0.1mm)	Nilaco	PT-351165
Platinum wire (φ0.5 mm)	Niaco	PT-351381
Scalpel	AsOne	8-3086-02 (Feather #11)
Scientific cMOS camera	Thorlabs	CC215MU
Sea salt	NAPQO	Instant Ocean
Stereo microscope	Olympus	SZX7
Stimulus isolater	AMPI	ISO-FLEX
Suction tube	Warner Instruments	ST-3, 64-1406
Temperature controller	Warner Instruments	TC-324C
Theta glass capillary	Sutter Instrument	BT-150-10
Tricaine (MS-222)	TCI	T0941
Upright microscope	Olympus	BX51WI