果蝇三龄幼虫神经肌肉连接的免疫电生理研究

Raffaella Klima; Giulia Romano; Monsurat Gbadamosi; Aram Megighian; Fabian   Feiguin

doi:10.21769/BioProtoc.3913

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Peer-reviewed

Immuno-electrophysiology on Neuromuscular Junctions of Drosophila Third Instar Larva

果蝇三龄幼虫神经肌肉连接的免疫电生理研究

RK Raffaella Klima

Giulia Romano

MG Monsurat Gbadamosi

AM Aram Megighian email

FF Fabian Feiguin email

发布: 2021年02月05日第11卷第3期 DOI: 10.21769/BioProtoc.3913 浏览次数: 3041

评审: Alexandros AlexandratosPradeep Kumar BhaskarFilipa Vaz

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Abstract

Alterations in synaptic transmission are critical early events in neuromuscular disorders. However, reliable methodologies to analyze the functional organization of the neuromuscular synapses are still needed. This manuscript provides a detailed protocol to analyze the molecular assembly of the neuromuscular synapses through immune-electrophysiology in Drosophila melanogaster. This technique allows the quantification of the molecular behavior of the neuromuscular synapses by correlating the structural configuration of the synaptic boutons with their electrical activity.

Keywords: Neuromuscular synapses (神经肌肉突触)

Synaptic boutons (突触束)

Neurological disorders (神经障碍)

Electrophysiology (电生理学)

Immunocytochemistry (免疫细胞化学)

Third instar larvae (三龄幼虫)

Drosophila melanogaster (黑腹果蝇)

Background

The functional organization of the neuromuscular junctions (NMJ) in Drosophila melanogaster third instar larva present prominent advantages to study the molecular mechanisms of synaptic formation and function (Feiguin et al., 2009; Godena et al., 2011; Romano et al., 2014 and 2015; Strah et al., 2020), which appears conserved in other species including humans. In that respect, the anatomical organization of the Drosophila NMJs is made of multiple synaptic boutons, formed at the final arborizations of the motor axons. The differentiation and maintenance of these structures are responsible for the innervation of the skeletal muscles and implicate the coordinated action of different molecules, specialized in the establishment of cell contacts and the machinery required to release, receive and integrate the neurotransmitter signaling. In addition, the powerful genetic tools developed in Drosophila together with the anatomical resolution of the nervous system to individual neurons, provide the rare opportunity to perform genome wide unbiased searches for molecular phenotypes in well distinguish cell tissues. Despite individual protocols to visualize the establishment of the neuromuscular synapses already exist in Drosophila (Sabeva and Bykhovskaia, 2017; Goel et al., 2019), in this manuscript (Figure 1) we provide a precise set of rules to bring together the simultaneous analysis of the molecular and physiological events that take place during the innervation of the skeletal muscles in vivo.

Figure 1. Synchronized steps in the functional analysis of the neuromuscular junctions

Materials and Reagents

Material and Reagents for NMJ immunohistochemistry
1. 0.5 ml tubes (Sarstedt, catalog number: 72.699 )
2. 2.0 ml tubes (Eppendorf, catalog number: 00 30120094 )
3. Whatmann qualitative filter paper (Sigma-Aldrich, catalog number: WHA1001917 )
4. Microscope slides Superfrost Plus (Thermo Fisher Scientific, catalog number: J1800AMNT )
5. Glass cover slips (Electron Microscopy Science, catalog number: 72210-10 )
6. Polystyrene Petri dish (94 mm, Greiner, catalog number: 633180 )
7. Small polystyrene dish (35 mm, CytoOne, catalog number: CC7672-3340 )
8. Drosophila melanogaster third instar larvae
  Note: Wild type strains like Canton S or Oregon R or specific lines can be obtained from Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/), from Kyoto stock Center DGRR (https://kyotofly.kit.jp/cgi-bin/stocks/index.cgi), from VDRC (https://stockcenter.vdrc.at/control/main) and from FlyORF (https://www.flyorf.ch/).
9. 0.7% agarose plates to keep selected larvae (tissue culture dishes 60 x 15 mm (Falcon, catalog number: 353004 ), Ultra pure agarose (Thermo Fischer, catalog number: 16500100 ). Plates should be stored, well wrapped in plastic bags in fridge at 4 °C
10. 16% paraformaldheyde (Electron Microscopy Science, catalog number: 15710 ), keep 500 μl aliquots in 2 ml tubes at -20 °C
11. Tween-20 (Sigma-Aldrich, catalog number: P9416 )
12. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C5080 )
13. HEPES (Sigma-Aldrich, catalog number: H3375 )
14. HEPES sodium salt (Sigma-Aldrich, catalog number: H7006 )
15. Magnesium Chloride solution (Sigma-Aldrich, catalog number: M1028 )
16. Potassium Chloride (KCl) (Sigma-Aldrich, catalog number: P3911 )
17. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
18. Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S6014 )
19. Sucrose (Sigma-Aldrich, catalog number: S1888 )
20. Trehalose (Sigma-Aldrich, catalog number: T9531 )
21. Normal Goat Serum (VectorLabs, catalog number: S-1000 ), keep aliquoted at -20 °C
22. AffiniPure Rabbit Anti-Horseradish Peroxidase (Jackson Immunoresearch, catalog number: 323-005-021 )
23. Cy3-Affinipure Goat Anti-HRP (Jackson Immunoresearch, catalog number: 123-165-021 )
24. Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Thermo Fisher Scientific, catalog number: A21428 ), store 4 °C aliquoted, light protected
25. Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, catalog number: A11008 ), store 4 °C aliquoted, light protected
26. Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher Scientific, catalog number: A21244 ), store 4 °C aliquoted, light protected
27. Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Thermo Fisher Scientific, catalog number: A21422 ), store 4 °C aliquoted, light protected
28. Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, catalog number: A11001 ), store 4 °C aliquoted, light protected
29. Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher Scientific, catalog number: A21235 ), store 4 °C aliquoted, light protected
30. Alexa Fluor Phalloidin 555 (Thermo Fisher Scientific, catalog number: A34055 ), store -20 °C, aliquoted, light protected
31. Alexa Fluor Phalloidin 350 (Thermo Fisher Scientific, catalog number: A22281 ), store -20 °C, aliquoted, light protected
32. Alexa Fluor Phalloidin 488 (Thermo Fisher Scientific, catalog number: A12379 ), store -20 °C, aliquoted, light protected
33. Slow Fade Gold (Thermo Fisher Scientific, catalog number: S36936 ), store -20 °C
34. Elastomer (Sylgard 184 curing agent elastomer, Down Corning, catalog number: DOWC6341655 )
35. Charcoal-Darco-100 mesh powder (Aldrich, catalog number: 242276 )
36. Sylgard dissection plates, both transparent and black (see Recipes)
37. HL-3-Ca²⁺free solution (see Recipes)
38. 4% PFA (see Recipes)
39. PBS 1× (see Recipes)
40. PBT (see Recipes)
41. 5% NGS (see Recipes)

Material and Reagents for NMJ electrophysiology
1. Silver wire (World Precision Instruments, catalog number: AGW2030 )
2. Flexible needle electrode Microfil (World Precision Instruments, catalog number: MF34G-5 )
3. Glass capillaries for intracellular microelectrodes (length 100 mm, inner diameter 0.86 mm, outer diameter 1.50 mm; Science Products, catalog number: GB150F-10 )
4. Glass capillaries for stimulating suction microelectrode (length 100 mm, inner diameter 1.05 mm, outer diameter 1.50 mm; Science Products, catalog number: GB150TF-10 )
5. Microelectrode holder with pressure port for suction microelectrodes (WPI, catalog number: MEH6SFW )
6. Microelectrode holder for intracellular microelectrodes (ALA Scientific Instruments, catalog number: BNC-PPH-0P-1.5 )
7. Iron (III) chloride (FeCl₃) (Sigma-Aldrich, catalog number: 451649 )
8. Silver chloride (AgCl) (Sigma-Aldrich, catalog number: 204382 )
9. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
11. Magnesium chloride, standard solution 1 M (MgCl₂) (Honeywell International, Fluka, catalog number: 63020 )
12. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C5080 )
13. Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: H1758 )
14. Potassium acetate (CH₃COOK) (Sigma-Aldrich, catalog number: P3542 )
15. HEPES (Sigma-Aldrich, catalog number: H3784 )
16. Threalose (Sigma-Aldrich, catalog number: T9531 )
17. Sucrose (Sigma-Aldrich, catalog number: S1888 )
18. Intracellular microelectrode filling solution (see Recipes)
19. Sylgard dissection plates (see Recipes)

Equipment

Equipment for NMJ Dissection
1. Stainless Steel Minutien Pins (FST, catalog number: 26002-10 )
2. One tweezer Dumont n.°5 student standard tip 0.1 × 0.06, for larvae and pin manipulation (FST, catalog number: 91150-20 )
3. Two tweezers Dumont n.°5 fine forceps tip 0.05 × 0.01 (FST, catalog number: 11254-20) for dissection
4. Spring Scissor 2.5 mm Blades Straight (FST, catalog number: 15000-08 )
5. Pipetman Classic P200 (Gilson, catalog number: F123601 )
6. Pipette Tip 200 (Sarstedt, catalog number: 70.760.002 )
7. Stereomicroscope for dissection (Leica, catalog number: MZ7.5 )
8. Goose neck 2 arms (Leica, catalog number: 30130021 )
9. Fluorescence stereomicroscope with UV light for larval selection if needed (Leica, catalog number: MZ12.5 )

Equipment for NMJ immunohistochemistry
1. Nutating mixer (LabNet, GyroMini, catalog number: S0500 )
2. Pipetman Classic P10 (Gilson, catalog number: F144802 )
3. Pipetman Classic P200 (Gilson, catalog number: F123601 )
4. Pipetman Classic P1000 (Gilson, catalog number: F123602 )
5. Pipette Tip 10 (Sarstedt, catalog number: 70.113 0)
6. Pipette Tip 200 (Sarstedt, catalog number: 70.760.002 )
7. Pipette Tip 1000 (Sarstedt, catalog number: 70.762.100 )
8. Confocal laser microscope (Zeiss, model: LSM880 )

Equipment for NMJ electrophysiology
Electrophysiology setup
1. Antivibration table (Vision IsoStation, Newport, https://www.newport.com.cn/)
2. Stereomicroscope for the electrophysiology setup (Leica Microsystem, model: Leica MZ125 , numeric aperture 0.8 with plan apochromatic objective 1.6×)
3. Hydraulic micromanipulator for intracellular recording electrode (NARISHIGE, model: MHW-103 , Three-axis Water Hydraulic Micromanipulator)
4. Stimulation electrode micromanipulators (Manual Micromanipulator, MARZHAUSER WETZLAR, model: MM 33 )
5. Faraday cage
6. Stimulator: S88 stimulator (Grass, Warwick, RI, USA)
7. Amplifier: intracellular bridge mode amplifier (Npi Electronic, model: BA-01X )
8. Pipette puller ( P-97 Flaming/Brown Micropipette Puller) (Sutter Instruments, model: P-97 )
9. A/D interface (National Instruments, model: PCI-6221 ) and computer compatible with the software
10. PC for online visualization of recorded signals and offline analysis of recorded traces

Software

ImageJ ( Wayne Rasband, NIH, https://imagej.nih.gov/)
Prism (GraphPad, https://www.graphpad.com/scientific-software/prism/)
WinEDR software (http://spider.science.strath.ac.uk/sipbs/software_ses.htm)

Procedure

English

中文翻译

文章信息

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如何引用

Klima, R., Romano, G., Gbadamos, M., Megighian, A. and Feiguin, F. (2021). Immuno-electrophysiology on Neuromuscular Junctions of Drosophila Third Instar Larva. Bio-protocol 11(3): e3913. DOI: 10.21769/BioProtoc.3913.