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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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Effect of Brefeldin  A (BFA) on quantal ACh release from growing Xenopus  axons. (A and B) Current  traces are examples of  whole-cell current recordings from the myocyte manipulated into contact with  the middle axonal segment  (top traces) or from myocyte  at the preformed synapse of  Xenopus axon (bottom  traces) 1 d after cell culture  preparation. Arrows indicate the onset of BFA application (10 μg/ml). After 5  min of recording, the whole-cell pipette was withdrawn  and recording from the same myocyte was performed again 1 h  after the onset of BFA application (B). Almost complete inhibition of quantal ACh secretion was observed at the middle axon  but not at the nerve terminal after BFA treatment. (C) Quantitative analysis of the effect of BFA on the quantal ACh secretion  from the axon. Each bar represents the average of 14–27 series of  experiments ± SEM. About a 30-fold decrease in the SSC frequency was observed at the middle axonal segment 1 h after BFA  treatment. The reduction in the SSC frequency found in the  spontaneously formed synapses after BFA treatment was not statistically significant (P > 0.05, t test). After a 30-min BFA wash  out, the frequency of secretion events at various axonal regions  was similar to that recorded in control cultures.
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Figure 9: Effect of Brefeldin A (BFA) on quantal ACh release from growing Xenopus axons. (A and B) Current traces are examples of whole-cell current recordings from the myocyte manipulated into contact with the middle axonal segment (top traces) or from myocyte at the preformed synapse of Xenopus axon (bottom traces) 1 d after cell culture preparation. Arrows indicate the onset of BFA application (10 μg/ml). After 5 min of recording, the whole-cell pipette was withdrawn and recording from the same myocyte was performed again 1 h after the onset of BFA application (B). Almost complete inhibition of quantal ACh secretion was observed at the middle axon but not at the nerve terminal after BFA treatment. (C) Quantitative analysis of the effect of BFA on the quantal ACh secretion from the axon. Each bar represents the average of 14–27 series of experiments ± SEM. About a 30-fold decrease in the SSC frequency was observed at the middle axonal segment 1 h after BFA treatment. The reduction in the SSC frequency found in the spontaneously formed synapses after BFA treatment was not statistically significant (P > 0.05, t test). After a 30-min BFA wash out, the frequency of secretion events at various axonal regions was similar to that recorded in control cultures.

Mentions: Generation of carrier vesicles from the intracellular membrane compartments requires GTP-binding proteins and coats (Rothman and Wieland, 1996; Schekman and Orci, 1996). In many cases, coat assembly is regulated by a small GTP-binding protein ARF1 (Donaldson et al., 1992). To test whether synaptic vesicle recycling at the preformed synapses and at the middle axon is mediated by ARF proteins, we treated neuronal cultures with Brefeldin A (BFA), a specific inhibitor of ARF1-mediated processes. 1 h after the onset of BFA treatment (10 μg/ml), the frequency of SSCs at the preformed synapses did not change significantly in comparison with control (untreated with BFA) neurons (Fig. 9). Surprisingly, neurotransmitter secretion in the middle segment of neurite was dramatically inhibited. The inhibition of secretory activity by BFA was completely reversible and is unlikely to reflect permanent damage to the neurons.


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

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

Effect of Brefeldin  A (BFA) on quantal ACh release from growing Xenopus  axons. (A and B) Current  traces are examples of  whole-cell current recordings from the myocyte manipulated into contact with  the middle axonal segment  (top traces) or from myocyte  at the preformed synapse of  Xenopus axon (bottom  traces) 1 d after cell culture  preparation. Arrows indicate the onset of BFA application (10 μg/ml). After 5  min of recording, the whole-cell pipette was withdrawn  and recording from the same myocyte was performed again 1 h  after the onset of BFA application (B). Almost complete inhibition of quantal ACh secretion was observed at the middle axon  but not at the nerve terminal after BFA treatment. (C) Quantitative analysis of the effect of BFA on the quantal ACh secretion  from the axon. Each bar represents the average of 14–27 series of  experiments ± SEM. About a 30-fold decrease in the SSC frequency was observed at the middle axonal segment 1 h after BFA  treatment. The reduction in the SSC frequency found in the  spontaneously formed synapses after BFA treatment was not statistically significant (P > 0.05, t test). After a 30-min BFA wash  out, the frequency of secretion events at various axonal regions  was similar to that recorded in control cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132923&req=5

Figure 9: Effect of Brefeldin A (BFA) on quantal ACh release from growing Xenopus axons. (A and B) Current traces are examples of whole-cell current recordings from the myocyte manipulated into contact with the middle axonal segment (top traces) or from myocyte at the preformed synapse of Xenopus axon (bottom traces) 1 d after cell culture preparation. Arrows indicate the onset of BFA application (10 μg/ml). After 5 min of recording, the whole-cell pipette was withdrawn and recording from the same myocyte was performed again 1 h after the onset of BFA application (B). Almost complete inhibition of quantal ACh secretion was observed at the middle axon but not at the nerve terminal after BFA treatment. (C) Quantitative analysis of the effect of BFA on the quantal ACh secretion from the axon. Each bar represents the average of 14–27 series of experiments ± SEM. About a 30-fold decrease in the SSC frequency was observed at the middle axonal segment 1 h after BFA treatment. The reduction in the SSC frequency found in the spontaneously formed synapses after BFA treatment was not statistically significant (P > 0.05, t test). After a 30-min BFA wash out, the frequency of secretion events at various axonal regions was similar to that recorded in control cultures.
Mentions: Generation of carrier vesicles from the intracellular membrane compartments requires GTP-binding proteins and coats (Rothman and Wieland, 1996; Schekman and Orci, 1996). In many cases, coat assembly is regulated by a small GTP-binding protein ARF1 (Donaldson et al., 1992). To test whether synaptic vesicle recycling at the preformed synapses and at the middle axon is mediated by ARF proteins, we treated neuronal cultures with Brefeldin A (BFA), a specific inhibitor of ARF1-mediated processes. 1 h after the onset of BFA treatment (10 μg/ml), the frequency of SSCs at the preformed synapses did not change significantly in comparison with control (untreated with BFA) neurons (Fig. 9). Surprisingly, neurotransmitter secretion in the middle segment of neurite was dramatically inhibited. The inhibition of secretory activity by BFA was completely reversible and is unlikely to reflect permanent damage to the neurons.

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