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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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Spontaneous release of ACh from Xenopus neurons.  (A) Schematic diagram or recording configuration. An isolated  Xenopus myocyte was detached from the substrate, clamped at  the resting membrane potential (−70 mV) using whole-cell patch  clamp technique, and then sequentially manipulated into contact  with three different sites along the axon. At each site the whole-cell patch clamp recording was performed for ∼5 min. (B) A representative example of membrane current recorded from a voltage-clamped myocyte. Downward deflections represent SSCs.  SSCs could be detected immediately after establishment of contact between the myocyte and the axon (horizontal black bars).  (C) Changes in the frequency and amplitude of the current  events with time after the onset of recording, normalized to the  values at the beginning of the recording. Data from eight recordings at preformed synapses (circles) and from 10 recordings at  the middle axonal segment (squares). Membrane current was  continuously recorded for a period of 35 min. In recordings from  the middle axon the data were collected immediately after establishment of contact between the myocyte and the axon. Notice  that the SSC frequency and SSC amplitude do not significantly  change during the period of recording.
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Figure 1: Spontaneous release of ACh from Xenopus neurons. (A) Schematic diagram or recording configuration. An isolated Xenopus myocyte was detached from the substrate, clamped at the resting membrane potential (−70 mV) using whole-cell patch clamp technique, and then sequentially manipulated into contact with three different sites along the axon. At each site the whole-cell patch clamp recording was performed for ∼5 min. (B) A representative example of membrane current recorded from a voltage-clamped myocyte. Downward deflections represent SSCs. SSCs could be detected immediately after establishment of contact between the myocyte and the axon (horizontal black bars). (C) Changes in the frequency and amplitude of the current events with time after the onset of recording, normalized to the values at the beginning of the recording. Data from eight recordings at preformed synapses (circles) and from 10 recordings at the middle axonal segment (squares). Membrane current was continuously recorded for a period of 35 min. In recordings from the middle axon the data were collected immediately after establishment of contact between the myocyte and the axon. Notice that the SSC frequency and SSC amplitude do not significantly change during the period of recording.

Mentions: Experiments were performed on 1-d-old nerve muscle cultures prepared from Xenopus embryos. Neurons and myocytes in culture formed contacts spontaneously. We will refer to these developing neuromuscular synapses as preformed synapses. Quantal release of ACh at the preformed synapses can be detected by the whole-cell voltage clamp recordings from the postsynaptic myocyte (Hamill et al., 1981). Individual spontaneous synaptic currents (SSCs) in recordings from myocytes reflect spontaneous exocytosis of ACh-containing synaptic vesicles and release of ACh quanta (Chow and Poo, 1985; Evers et al., 1989). To detect the release of ACh in growing axons we chose axon-bearing neurons that were free of contact with other cells. An isolated Xenopus myocyte was detached from the substrate, voltage clamped at the resting membrane potential (−70 mV) using whole-cell patch clamp technique, and then manipulated into contact with the axonal shaft (Fig. 1 A). Recordings of the membrane currents in the myocyte using a whole-cell voltage clamp technique revealed fast inward currents (Fig. 1 A). These currents could be detected immediately after manipulation of the myocyte into contact with the neurite, and the SSC frequency did not change during the 30-min period of recordings (Fig. 1 B). Hence, myocytes appear to serve as passive detectors, rather than inducers, of exocytic events (Girod et al., 1995; Ninomiya et al., 1997; Antonov et al., 1998). In our previous study (Antonov et al., 1998) we showed that release of ACh packets can be detected throughout the whole neuronal surface. We characterized the distribution of ACh secretion along the growing axons and demonstrated that the frequency of SSCs displayed a proximodistal gradient with a higher level of activity at the distal axonal region. Moreover, the parameters of individual SSCs (rise time, decay time, frequency, and amplitude) recorded from different axonal segments were found to be very similar (Antonov et al., 1998). In this study we focused on ACh secretion at two neuronal regions: the middle axonal segment in naive (free of contact with other cells) neurons and the nerve terminal in the preformed neuromuscular synapses.


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 from Xenopus neurons.  (A) Schematic diagram or recording configuration. An isolated  Xenopus myocyte was detached from the substrate, clamped at  the resting membrane potential (−70 mV) using whole-cell patch  clamp technique, and then sequentially manipulated into contact  with three different sites along the axon. At each site the whole-cell patch clamp recording was performed for ∼5 min. (B) A representative example of membrane current recorded from a voltage-clamped myocyte. Downward deflections represent SSCs.  SSCs could be detected immediately after establishment of contact between the myocyte and the axon (horizontal black bars).  (C) Changes in the frequency and amplitude of the current  events with time after the onset of recording, normalized to the  values at the beginning of the recording. Data from eight recordings at preformed synapses (circles) and from 10 recordings at  the middle axonal segment (squares). Membrane current was  continuously recorded for a period of 35 min. In recordings from  the middle axon the data were collected immediately after establishment of contact between the myocyte and the axon. Notice  that the SSC frequency and SSC amplitude do not significantly  change during the period of recording.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132923&req=5

Figure 1: Spontaneous release of ACh from Xenopus neurons. (A) Schematic diagram or recording configuration. An isolated Xenopus myocyte was detached from the substrate, clamped at the resting membrane potential (−70 mV) using whole-cell patch clamp technique, and then sequentially manipulated into contact with three different sites along the axon. At each site the whole-cell patch clamp recording was performed for ∼5 min. (B) A representative example of membrane current recorded from a voltage-clamped myocyte. Downward deflections represent SSCs. SSCs could be detected immediately after establishment of contact between the myocyte and the axon (horizontal black bars). (C) Changes in the frequency and amplitude of the current events with time after the onset of recording, normalized to the values at the beginning of the recording. Data from eight recordings at preformed synapses (circles) and from 10 recordings at the middle axonal segment (squares). Membrane current was continuously recorded for a period of 35 min. In recordings from the middle axon the data were collected immediately after establishment of contact between the myocyte and the axon. Notice that the SSC frequency and SSC amplitude do not significantly change during the period of recording.
Mentions: Experiments were performed on 1-d-old nerve muscle cultures prepared from Xenopus embryos. Neurons and myocytes in culture formed contacts spontaneously. We will refer to these developing neuromuscular synapses as preformed synapses. Quantal release of ACh at the preformed synapses can be detected by the whole-cell voltage clamp recordings from the postsynaptic myocyte (Hamill et al., 1981). Individual spontaneous synaptic currents (SSCs) in recordings from myocytes reflect spontaneous exocytosis of ACh-containing synaptic vesicles and release of ACh quanta (Chow and Poo, 1985; Evers et al., 1989). To detect the release of ACh in growing axons we chose axon-bearing neurons that were free of contact with other cells. An isolated Xenopus myocyte was detached from the substrate, voltage clamped at the resting membrane potential (−70 mV) using whole-cell patch clamp technique, and then manipulated into contact with the axonal shaft (Fig. 1 A). Recordings of the membrane currents in the myocyte using a whole-cell voltage clamp technique revealed fast inward currents (Fig. 1 A). These currents could be detected immediately after manipulation of the myocyte into contact with the neurite, and the SSC frequency did not change during the 30-min period of recordings (Fig. 1 B). Hence, myocytes appear to serve as passive detectors, rather than inducers, of exocytic events (Girod et al., 1995; Ninomiya et al., 1997; Antonov et al., 1998). In our previous study (Antonov et al., 1998) we showed that release of ACh packets can be detected throughout the whole neuronal surface. We characterized the distribution of ACh secretion along the growing axons and demonstrated that the frequency of SSCs displayed a proximodistal gradient with a higher level of activity at the distal axonal region. Moreover, the parameters of individual SSCs (rise time, decay time, frequency, and amplitude) recorded from different axonal segments were found to be very similar (Antonov et al., 1998). In this study we focused on ACh secretion at two neuronal regions: the middle axonal segment in naive (free of contact with other cells) neurons and the nerve terminal in the preformed neuromuscular synapses.

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