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Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis.

Zakharenko S, Chang S, O'Donoghue M, Popov SV - J. Cell Biol. (1999)

Bottom Line: We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar.These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion.Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics M/C 901, University of Illinois, Chicago, Illinois 60612, USA.

ABSTRACT
In mature neurons, synaptic vesicles continuously recycle within the presynaptic nerve terminal. In developing axons which are free of contact with a postsynaptic target, constitutive membrane recycling is not localized to the nerve terminal; instead, plasma membrane components undergo cycles of exoendocytosis throughout the whole axonal surface (Matteoli et al., 1992; Kraszewski et al., 1995). Moreover, in growing Xenopus spinal cord neurons in culture, acetylcholine (ACh) is spontaneously secreted in the quantal fashion along the axonal shaft (Evers et al., 1989; Antonov et al., 1998). Here we demonstrate that in Xenopus neurons ACh secretion is mediated by vesicles which recycle locally within the axon. Similar to neurotransmitter release at the presynaptic nerve terminal, ACh secretion along the axon could be elicited by the action potential or by hypertonic solutions. We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar. These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion. Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface. Maturation of this machinery in the process of synaptic development would improve the fidelity of synaptic transmission during high-frequency stimulation of the presynaptic cell.

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(A) DIC (top) and fluorescence images of Xenopus  neuron after staining with FM1-43, and after superfusion with  60 mM KCl in Ca2+-free medium followed by superfusion with  solution containing 60 mM KCl and 2 mM Ca2+. Staining revealed a characteristic array of fluorescent spots, which are likely  to represent clusters of synaptic vesicles. FM1-43–stained organelles were rapidly destained upon KCl-induced depolarization in the medium containing 2 mM Ca2+ but not in the Ca2+-free medium. Destaining could be detected both at the middle  axonal segment and at the growth cone region. (B) The average  brightness of the fluorescent spots during destaining. For each  cell the intensity was normalized to that at the onset of depolarization before averaging. Data from 80 fluorescent spots in eight  different cells. Destaining at the middle axonal segment (open  circles) and at the growth cone region (filled squares) occurred  with a similar kinetics. No statistically significant destaining was  observed when perfusion medium contained 5 mM EGTA  (Ca2+-free medium).
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Figure 5: (A) DIC (top) and fluorescence images of Xenopus neuron after staining with FM1-43, and after superfusion with 60 mM KCl in Ca2+-free medium followed by superfusion with solution containing 60 mM KCl and 2 mM Ca2+. Staining revealed a characteristic array of fluorescent spots, which are likely to represent clusters of synaptic vesicles. FM1-43–stained organelles were rapidly destained upon KCl-induced depolarization in the medium containing 2 mM Ca2+ but not in the Ca2+-free medium. Destaining could be detected both at the middle axonal segment and at the growth cone region. (B) The average brightness of the fluorescent spots during destaining. For each cell the intensity was normalized to that at the onset of depolarization before averaging. Data from 80 fluorescent spots in eight different cells. Destaining at the middle axonal segment (open circles) and at the growth cone region (filled squares) occurred with a similar kinetics. No statistically significant destaining was observed when perfusion medium contained 5 mM EGTA (Ca2+-free medium).

Mentions: Endocytic membrane compartments in the neuron can be stained with fluorescent membrane dye FM1-43. Depolarization-induced destaining of the neurons is believed to reflect the Ca2+-dependent fusion of FM1-43–labeled synaptic vesicles with the plasma membrane and release of the dye into the extracellular medium (Betz and Bewick, 1992; Ryan et al., 1993). In agreement with previously published data (Kraszewski et al., 1995; Dai and Peng, 1996a) we found that after incubation with FM1-43, staining of neurites was not uniform. Occasionally individual fluorescent spots could be resolved (Fig. 5). These spots are likely to represent clusters of synaptic vesicles (Kraszewski et al., 1995). Repeated images were acquired while the neuron was superfused with a 60 mM KCl. The average brightness of the spots rapidly decreased with time during superfusion. No destaining of FM1-43–labeled organelles was observed when superfusion with 60 mM KCl was done in the Ca2+-free medium. This result suggests that, similar to the nerve terminal, exocytosis of synaptic vesicles along the axon is Ca2+-dependent.


Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis.

Zakharenko S, Chang S, O'Donoghue M, Popov SV - J. Cell Biol. (1999)

(A) DIC (top) and fluorescence images of Xenopus  neuron after staining with FM1-43, and after superfusion with  60 mM KCl in Ca2+-free medium followed by superfusion with  solution containing 60 mM KCl and 2 mM Ca2+. Staining revealed a characteristic array of fluorescent spots, which are likely  to represent clusters of synaptic vesicles. FM1-43–stained organelles were rapidly destained upon KCl-induced depolarization in the medium containing 2 mM Ca2+ but not in the Ca2+-free medium. Destaining could be detected both at the middle  axonal segment and at the growth cone region. (B) The average  brightness of the fluorescent spots during destaining. For each  cell the intensity was normalized to that at the onset of depolarization before averaging. Data from 80 fluorescent spots in eight  different cells. Destaining at the middle axonal segment (open  circles) and at the growth cone region (filled squares) occurred  with a similar kinetics. No statistically significant destaining was  observed when perfusion medium contained 5 mM EGTA  (Ca2+-free medium).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132923&req=5

Figure 5: (A) DIC (top) and fluorescence images of Xenopus neuron after staining with FM1-43, and after superfusion with 60 mM KCl in Ca2+-free medium followed by superfusion with solution containing 60 mM KCl and 2 mM Ca2+. Staining revealed a characteristic array of fluorescent spots, which are likely to represent clusters of synaptic vesicles. FM1-43–stained organelles were rapidly destained upon KCl-induced depolarization in the medium containing 2 mM Ca2+ but not in the Ca2+-free medium. Destaining could be detected both at the middle axonal segment and at the growth cone region. (B) The average brightness of the fluorescent spots during destaining. For each cell the intensity was normalized to that at the onset of depolarization before averaging. Data from 80 fluorescent spots in eight different cells. Destaining at the middle axonal segment (open circles) and at the growth cone region (filled squares) occurred with a similar kinetics. No statistically significant destaining was observed when perfusion medium contained 5 mM EGTA (Ca2+-free medium).
Mentions: Endocytic membrane compartments in the neuron can be stained with fluorescent membrane dye FM1-43. Depolarization-induced destaining of the neurons is believed to reflect the Ca2+-dependent fusion of FM1-43–labeled synaptic vesicles with the plasma membrane and release of the dye into the extracellular medium (Betz and Bewick, 1992; Ryan et al., 1993). In agreement with previously published data (Kraszewski et al., 1995; Dai and Peng, 1996a) we found that after incubation with FM1-43, staining of neurites was not uniform. Occasionally individual fluorescent spots could be resolved (Fig. 5). These spots are likely to represent clusters of synaptic vesicles (Kraszewski et al., 1995). Repeated images were acquired while the neuron was superfused with a 60 mM KCl. The average brightness of the spots rapidly decreased with time during superfusion. No destaining of FM1-43–labeled organelles was observed when superfusion with 60 mM KCl was done in the Ca2+-free medium. This result suggests that, similar to the nerve terminal, exocytosis of synaptic vesicles along the axon is Ca2+-dependent.

Bottom Line: We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar.These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion.Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics M/C 901, University of Illinois, Chicago, Illinois 60612, USA.

ABSTRACT
In mature neurons, synaptic vesicles continuously recycle within the presynaptic nerve terminal. In developing axons which are free of contact with a postsynaptic target, constitutive membrane recycling is not localized to the nerve terminal; instead, plasma membrane components undergo cycles of exoendocytosis throughout the whole axonal surface (Matteoli et al., 1992; Kraszewski et al., 1995). Moreover, in growing Xenopus spinal cord neurons in culture, acetylcholine (ACh) is spontaneously secreted in the quantal fashion along the axonal shaft (Evers et al., 1989; Antonov et al., 1998). Here we demonstrate that in Xenopus neurons ACh secretion is mediated by vesicles which recycle locally within the axon. Similar to neurotransmitter release at the presynaptic nerve terminal, ACh secretion along the axon could be elicited by the action potential or by hypertonic solutions. We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar. These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion. Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface. Maturation of this machinery in the process of synaptic development would improve the fidelity of synaptic transmission during high-frequency stimulation of the presynaptic cell.

Show MeSH
Related in: MedlinePlus