Use of fluorescent probes to follow membrane traffic in nerve terminals

International Symposium: Biological Applications of Confocal Microscopy • Braz J Med Biol Res 31 (11) • Nov 1998 • https://doi.org/10.1590/S0100-879X1998001100018 copy

Use of fluorescent probes to follow membrane traffic in nerve terminals

Authorship SCIMAGO INSTITUTIONS RANKINGS

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.

laser scanning confocal microscopy; FM1-43; exocytosis; synaptic vesicles; endocytosis

Authorship

C. Guatimosim

Universidade Federal de Minas Gerais, Laboratório de Neurofarmacologia, Departamento de Farmacologia, Instituto de Ciências Biológicas, Minas Gerais, BrazilUniversidade Federal de Minas GeraisBrazilMinas Gerais, BrazilUniversidade Federal de Minas Gerais, Laboratório de Neurofarmacologia, Departamento de Farmacologia, Instituto de Ciências Biológicas, Minas Gerais, Brazil

M.A. Romano-Silva

M.V. Gomez

M.A.M. Prado

SCIMAGO INSTITUTIONS RANKINGS

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Figure 1
- Staining and destaining of frog motor nerve terminals labeled with FM1-43.
A, Motor nerve terminals from frog cutaneous pectoris muscle were labeled with FM1-43 during electrical stimulation of the nerve (20 Hz, 5 min). After a washing period, FM1-43-stained terminals were selected using a Zeiss fluorescence microscope (axiovert 100) with a 40-X water immersion objective (1.2 NA). The nerve terminals were imaged using an MRC 1024-BioRad argon UV laser scanning confocal microscope coupled to the Zeiss microscope. The preparation was excited at 488 nm and light emitted above 515 nm was collected for analysis. This figure shows a low magnification confocal image obtained after reconstruction of a series of Z-sections (0.5 µm apart, iris at 1.7). Note the presence of several fluorescent puncta, each of which corresponds to a cluster of synaptic vesicles labeled with FM1-43. Bar, 30 µm.
B, FM1-43-labeled motor nerve terminals such as those described in A were electrically stimulated through the nerve (20 Hz, 5 min) in dye-free medium. The fluorescent puncta destained progressively during stimulation and the loss of fluorescence was quantified as mean fluorescence intensity in 256 levels of grey. The dashed curve represents the mean intensity of fluorescence destaining of 17 fluorescent puncta and the solid line corresponds to the fitting of the destaining curve by a single exponential equation (fluorescence (t) = Ae^-t/t + C). The destaining curve presented a t = 58 ± 4 s (data from Ref. 32).
C, Rat cerebrocortical synaptosomes were labeled with FM1-43 according to Ref. 27. After labeling, the synaptosomes were washed in dye-free medium and transferred to a fluorimeter cuvette. Fluorescence measurements were performed with a PTI spectrofluorimeter, at 488 nm (excitation) and 560 nm (emission). FM1-43-labeled synaptosomes were destained under depolarization with 60 mM KCl. The destaining curve (dashed line) represents the mean of twelve independent experiments and is described by a double exponential equation (fluorescence (t) = A₁e^-t/t1 + A₂e^-t/t2 + C), with time constants of 8 ± 1 s and 129 ± 9 s for the first and second components, respectively. The solid line represents the fitting of the FM1-43-destaining curve for synaptosomes.

Figure 1 - Staining and destaining of frog motor nerve terminals labeled with FM1-43.
A, Motor nerve terminals from frog cutaneous pectoris muscle were labeled with FM1-43 during electrical stimulation of the nerve (20 Hz, 5 min). After a washing period, FM1-43-stained terminals were selected using a Zeiss fluorescence microscope (axiovert 100) with a 40-X water immersion objective (1.2 NA). The nerve terminals were imaged using an MRC 1024-BioRad argon UV laser scanning confocal microscope coupled to the Zeiss microscope. The preparation was excited at 488 nm and light emitted above 515 nm was collected for analysis. This figure shows a low magnification confocal image obtained after reconstruction of a series of Z-sections (0.5 µm apart, iris at 1.7). Note the presence of several fluorescent puncta, each of which corresponds to a cluster of synaptic vesicles labeled with FM1-43. Bar, 30 µm.
B, FM1-43-labeled motor nerve terminals such as those described in A were electrically stimulated through the nerve (20 Hz, 5 min) in dye-free medium. The fluorescent puncta destained progressively during stimulation and the loss of fluorescence was quantified as mean fluorescence intensity in 256 levels of grey. The dashed curve represents the mean intensity of fluorescence destaining of 17 fluorescent puncta and the solid line corresponds to the fitting of the destaining curve by a single exponential equation (fluorescence (t) = Ae^-t/t + C). The destaining curve presented a t = 58 ± 4 s (data from Ref. 32).
C, Rat cerebrocortical synaptosomes were labeled with FM1-43 according to Ref. 27. After labeling, the synaptosomes were washed in dye-free medium and transferred to a fluorimeter cuvette. Fluorescence measurements were performed with a PTI spectrofluorimeter, at 488 nm (excitation) and 560 nm (emission). FM1-43-labeled synaptosomes were destained under depolarization with 60 mM KCl. The destaining curve (dashed line) represents the mean of twelve independent experiments and is described by a double exponential equation (fluorescence (t) = A₁e^-t/t1 + A₂e^-t/t2 + C), with time constants of 8 ± 1 s and 129 ± 9 s for the first and second components, respectively. The solid line represents the fitting of the FM1-43-destaining curve for synaptosomes.

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Use of fluorescent probes to follow membrane traffic in nerve terminals

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