Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Summary

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Abstract

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.

Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon's start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Introduction

The modulation of synaptic function contributes to the remarkable ability of the nervous system to precisely respond or adapt to changing environmental stimuli. Adjusting the presynaptic vesicle release probability is one way of controlling synaptic strength¹. Synaptic vesicle release takes place at the Active Zone (AZ), a specialized region of the presynaptic membrane². The AZ is characterized by a cassette of specific proteins^3,4. Most proteins contributing to AZ assembly are highly conserved in nematodes, insects and mammals⁵. Recent st....

Protocol

The experimental procedures described in this protocol involve exclusively work with Drosophila and are not subjected to animal welfare laws in Germany and Japan.

1. Light Exposure Conditions

1. Prepare flies that carry sensless-flippase (sens-flp) and bruchpilot-FRT-STOP-FRT-gfp (brp-FSF-gfp)¹⁵ for visualizing Brp-GFP in photoreceptor R8 neurons (R8s). The genomic locus encoding brp within a Bacterial Artificial Chromosome (BAC) was modified to generate this brp-FSF-gfp construct. In the flies carrying this modified BAC the expression of Brp-GFP....

Results

The compound eye of Drosophila comprises ~780 ommatidia, each containing eight types of photoreceptors (R1-8). R7 and R8 project their axons to the second optic ganglion, the medulla, where they form synapses in layers M6 and M3, respectively²⁶. To investigate the effect of prolonged exposure to light on the molecular composition of photoreceptor R8 active zones, we took advantage of the STaR method¹⁵. Endogenous expression levels o.......

Discussion

In this study, we showed how to prepare the light conditions to expose flies to an equal light intensity. We quantified not only the number of a synaptic marker puncta but could also spatially resolve the density of synapses along axons and measure the delocalization level of the marker protein in cytoplasmic areas. These three assessments allow us to evaluate the details of synaptic dynamics at single neuron level in different environmental conditions. Our protocol can be adapted to different synaptic proteins but also .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vial	Hightech, Japan	MKC-20
Plug	Thermo Fisher Sciehtific, USA	AS-275
Customized transparent rack made of acrylic resin	Shin-Shin Corporation, Japan		a height of 41 cm, a base of 21 cm, a thickness of 4 cm and a height of 13 cm for each step
Cool incubator	MITSUBISHI ELECTRIC, Japan	CN-40A
LED panel	MISUMI, Japan	LEDXC170-W
Digital light meter	CEM	DT-1301
Fly pad	Tokken, Japan	TK-HA03-S
Petri dish (35 x 10 mm)	Greiner Bio-One International, Germany	627102
PBS tablet	Takara, Japan	T900
Triton X-100	Wako, Japan	160-24751
Dumont #5 Forceps	Fine Science Tools	11251-20
1.5 mL tube	Sarstedt, Germany	A. 152X
Formaldehyde 16%	NEM, Japan	3152
Pipetman P-200	Gilson	F123601
Pipetman P-20	Gilson	F123600
Pipetman P-2	Gilson	F144801
anti-chaoptin antibody	DSHB	24B10
Alexa568-conjugated anti-mouse antibody	Life Technologies	A-11031
VECTASHIELD Mounting Medium	Vector Laboratories, Inc.	H-1000
Microscope slide (76 x 26 mm)	Thermo Fisher Scientific Gerhard Menzel B.V. & Co. KG, Germany
Coverslip (18 x 18 mm, 0.17 mm)	Zeiss, Germany	474030-9000-000
Industrial Microscopes	Olympus, Japan	SZ61-C-SET
Stereo Microscope Lighting	Olympus, Japan	KL 1600 LED
confocal microscopy	Zeiss, Germany	LSM780
Imaris	Bitplane, Switzerland		Version 7.6.4 or above
Matlab	The MathWorks, Inc., USA
Excel for Mac	Microsoft