Limits...
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.

Show MeSH

Related in: MedlinePlus

Spontaneous release of ACh in  isolated Xenopus neurons is mediated by  the local recycling of ACh-containing vesicles along the axon. (A) Schematic diagram of recording configuration. Myocyte  (M) was manipulated into contact with the  middle axonal segment 1 d after cell culture preparation. Whole-cell configuration  was established ∼1–2 min after manipulation. Patch clamp recordings from the myocytes were performed for ∼30 min. The  neurons chosen for experiments were free  of contact with other cells and had a single  axon ∼300–500 μm in length. (B) The  trace is a representative example of membrane current recorded from whole-cell  voltage-clamped myocyte (Vh = −70 mV)  manipulated into contact with the middle  axonal segment. Downward deflections  represent SSCs. SSCs could be detected  immediately after establishment of whole-cell configuration (start of the recording).  Nocodazole (5 μg/ml, arrow) was applied  to the bath ∼5 min after the start of recording. (C) Representative fluorescent  images of Xenopus neurons loaded with  Cy3-Rhodamine before (top) and after  (bottom) detergent extraction in a microtubule-stabilizing buffer. In control cells  (left panels) the bulk of tubulin was retained in the neuron after extraction. In neurons treated for 30 min with nocodazole (5 μg/ml, right panels), most of the tubulin was removed during the extraction procedure. Similar results were obtained in experiments performed on 10 control neurons and on 10 nocodazole-treated neurons. (D) Nocodazole treatment arrests axonal growth. Representative differential interference contrast (DIC)  images of neurite 1 d after cell culture preparation. Numbers indicate time in minutes. Nocodazole (5 μg/ml) was applied 30 min after  the start of the experiment. Within 30 min after nocodazole application, axonal elongation slowed down and the growth cone retracted.  Similar results were observed in nine different experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132923&req=5

Figure 2: Spontaneous release of ACh in isolated Xenopus neurons is mediated by the local recycling of ACh-containing vesicles along the axon. (A) Schematic diagram of recording configuration. Myocyte (M) was manipulated into contact with the middle axonal segment 1 d after cell culture preparation. Whole-cell configuration was established ∼1–2 min after manipulation. Patch clamp recordings from the myocytes were performed for ∼30 min. The neurons chosen for experiments were free of contact with other cells and had a single axon ∼300–500 μm in length. (B) The trace is a representative example of membrane current recorded from whole-cell voltage-clamped myocyte (Vh = −70 mV) manipulated into contact with the middle axonal segment. Downward deflections represent SSCs. SSCs could be detected immediately after establishment of whole-cell configuration (start of the recording). Nocodazole (5 μg/ml, arrow) was applied to the bath ∼5 min after the start of recording. (C) Representative fluorescent images of Xenopus neurons loaded with Cy3-Rhodamine before (top) and after (bottom) detergent extraction in a microtubule-stabilizing buffer. In control cells (left panels) the bulk of tubulin was retained in the neuron after extraction. In neurons treated for 30 min with nocodazole (5 μg/ml, right panels), most of the tubulin was removed during the extraction procedure. Similar results were obtained in experiments performed on 10 control neurons and on 10 nocodazole-treated neurons. (D) Nocodazole treatment arrests axonal growth. Representative differential interference contrast (DIC) images of neurite 1 d after cell culture preparation. Numbers indicate time in minutes. Nocodazole (5 μg/ml) was applied 30 min after the start of the experiment. Within 30 min after nocodazole application, axonal elongation slowed down and the growth cone retracted. Similar results were observed in nine different experiments.

Mentions: The vesicles involved in spontaneous ACh secretion along the axon may recycle locally within the axon, similar to synaptic vesicles in the nerve terminal. Alternatively, these vesicles may be directly transported from the soma via a constitutive biosynthetic pathway (Nakata et al., 1998). In the latter scenario, the cell body-derived vesicles would be expected to carry the molecules of the ACh transporter to produce a detectable SSC upon their exocytosis (Song et al., 1997). To distinguish between the two possibilities, we treated neuronal cultures with 5 μg/ml nocodazole. This treatment resulted in the loss of axonal microtubules (Fig. 2 C), rapidly arrested axonal growth (Fig. 2 D), and inhibited transport of mitochondria along the axon (Fig. 3). Therefore, the treatment with nocodazole is expected to disrupt the delivery of cell body-derived vesicles to the growing axon. However, spontaneous neurotransmitter secretion persisted after nocodazole application both along the axon (Fig. 2 B) and at the preformed synapses (data not shown). Moreover, the disruption of axonal microtubules resulted in a significant increase in the SSC frequency at the middle axonal segment (detailed quantitative analysis of the effects of axonal microtubules on neurotransmitter secretion will be presented elsewhere). These results strongly suggest that constitutive ACh secretion along the axon is not directly related to the exocytosis of cell body-derived vesicles. Instead, ACh secretion is likely to be mediated by a local exoendocytic recycling of ACh-containing vesicles.


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

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

Spontaneous release of ACh in  isolated Xenopus neurons is mediated by  the local recycling of ACh-containing vesicles along the axon. (A) Schematic diagram of recording configuration. Myocyte  (M) was manipulated into contact with the  middle axonal segment 1 d after cell culture preparation. Whole-cell configuration  was established ∼1–2 min after manipulation. Patch clamp recordings from the myocytes were performed for ∼30 min. The  neurons chosen for experiments were free  of contact with other cells and had a single  axon ∼300–500 μm in length. (B) The  trace is a representative example of membrane current recorded from whole-cell  voltage-clamped myocyte (Vh = −70 mV)  manipulated into contact with the middle  axonal segment. Downward deflections  represent SSCs. SSCs could be detected  immediately after establishment of whole-cell configuration (start of the recording).  Nocodazole (5 μg/ml, arrow) was applied  to the bath ∼5 min after the start of recording. (C) Representative fluorescent  images of Xenopus neurons loaded with  Cy3-Rhodamine before (top) and after  (bottom) detergent extraction in a microtubule-stabilizing buffer. In control cells  (left panels) the bulk of tubulin was retained in the neuron after extraction. In neurons treated for 30 min with nocodazole (5 μg/ml, right panels), most of the tubulin was removed during the extraction procedure. Similar results were obtained in experiments performed on 10 control neurons and on 10 nocodazole-treated neurons. (D) Nocodazole treatment arrests axonal growth. Representative differential interference contrast (DIC)  images of neurite 1 d after cell culture preparation. Numbers indicate time in minutes. Nocodazole (5 μg/ml) was applied 30 min after  the start of the experiment. Within 30 min after nocodazole application, axonal elongation slowed down and the growth cone retracted.  Similar results were observed in nine different experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Spontaneous release of ACh in isolated Xenopus neurons is mediated by the local recycling of ACh-containing vesicles along the axon. (A) Schematic diagram of recording configuration. Myocyte (M) was manipulated into contact with the middle axonal segment 1 d after cell culture preparation. Whole-cell configuration was established ∼1–2 min after manipulation. Patch clamp recordings from the myocytes were performed for ∼30 min. The neurons chosen for experiments were free of contact with other cells and had a single axon ∼300–500 μm in length. (B) The trace is a representative example of membrane current recorded from whole-cell voltage-clamped myocyte (Vh = −70 mV) manipulated into contact with the middle axonal segment. Downward deflections represent SSCs. SSCs could be detected immediately after establishment of whole-cell configuration (start of the recording). Nocodazole (5 μg/ml, arrow) was applied to the bath ∼5 min after the start of recording. (C) Representative fluorescent images of Xenopus neurons loaded with Cy3-Rhodamine before (top) and after (bottom) detergent extraction in a microtubule-stabilizing buffer. In control cells (left panels) the bulk of tubulin was retained in the neuron after extraction. In neurons treated for 30 min with nocodazole (5 μg/ml, right panels), most of the tubulin was removed during the extraction procedure. Similar results were obtained in experiments performed on 10 control neurons and on 10 nocodazole-treated neurons. (D) Nocodazole treatment arrests axonal growth. Representative differential interference contrast (DIC) images of neurite 1 d after cell culture preparation. Numbers indicate time in minutes. Nocodazole (5 μg/ml) was applied 30 min after the start of the experiment. Within 30 min after nocodazole application, axonal elongation slowed down and the growth cone retracted. Similar results were observed in nine different experiments.
Mentions: The vesicles involved in spontaneous ACh secretion along the axon may recycle locally within the axon, similar to synaptic vesicles in the nerve terminal. Alternatively, these vesicles may be directly transported from the soma via a constitutive biosynthetic pathway (Nakata et al., 1998). In the latter scenario, the cell body-derived vesicles would be expected to carry the molecules of the ACh transporter to produce a detectable SSC upon their exocytosis (Song et al., 1997). To distinguish between the two possibilities, we treated neuronal cultures with 5 μg/ml nocodazole. This treatment resulted in the loss of axonal microtubules (Fig. 2 C), rapidly arrested axonal growth (Fig. 2 D), and inhibited transport of mitochondria along the axon (Fig. 3). Therefore, the treatment with nocodazole is expected to disrupt the delivery of cell body-derived vesicles to the growing axon. However, spontaneous neurotransmitter secretion persisted after nocodazole application both along the axon (Fig. 2 B) and at the preformed synapses (data not shown). Moreover, the disruption of axonal microtubules resulted in a significant increase in the SSC frequency at the middle axonal segment (detailed quantitative analysis of the effects of axonal microtubules on neurotransmitter secretion will be presented elsewhere). These results strongly suggest that constitutive ACh secretion along the axon is not directly related to the exocytosis of cell body-derived vesicles. Instead, ACh secretion is likely to be mediated by a local exoendocytic recycling of ACh-containing vesicles.

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