Chang S and Popov SV (1999), Long-range signaling within growing neurites me...

XB-ART-13291

Proc Natl Acad Sci U S A 1999 Mar 30;967:4095-100. doi: 10.1073/pnas.96.7.4095.

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Long-range signaling within growing neurites mediated by neurotrophin-3.

Chang S , Popov SV .

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In addition to well established trophic functions, neurotrophins acutely affect neurotransmitter secretion from the presynaptic nerve terminal, influence synaptic development, and may serve as selective retrograde messengers that regulate synaptic efficacy. The crucial question related to the mechanisms of neurotrophin-mediated signaling is whether acute effects of neurotrophins are spatially restricted to the activated synapses. Here we have used a local perfusion technique for local delivery of neurotrophin-3 (NT-3) to various regions of developing Xenopus embryo neurons in culture. Within minutes after a focal exposure of a soma or a small ( approximately 30 micrometer in length) axonal segment to NT-3, we observed an increase in the spontaneous neurotransmitter secretion from the presynaptic nerve terminals located approximately 300-400 micrometer away from the site of NT-3 application. Secretory activity along the axonal shaft was not affected. Our findings suggest that the NT-3-mediated signal may rapidly travel through neuronal cytoplasm over unexpectedly long distances and modulate neurotransmitter release specifically at the presynaptic nerve terminals.

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Species referenced: Xenopus
Genes referenced: bdnf ntf3 ntrk3
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	Figure 1 Acute effect of NT-3 application on SSC frequency at different axonal segments. (A) Schematic diagram of recording configurations. Whole-cell patch-clamp recordings were performed from the myocytes (M) at the spontaneous formed (“preformed”) synapses and from myocytes manipulated into contact with the growth cone region, the middle axonal segment, and the soma of isolated neurons 1 day after cell culture preparation. The neurons chosen for manipulation experiments were free of contact with other cells and had a single axon ≈300–400 μm in length. (B) Membrane currents recorded from the myocytes for a 3-min period before NT-3 application (“control”) and for a period of 15–18 min after the onset of NT-3 (50 ng/ml) treatment. Downward spikes are inward currents reflecting spontaneous ACh secretion from the neuron. (C) Changes in the SSC frequency with time after the bath application of NT-3 (marked by arrow). The mean SSC frequency was calculated for 2-min intervals and normalized to the mean SSC frequency for a 5-min period before NT-3 application. Each data point represents the mean ± SEM of 5–15 experiments. ∗, significantly different from control values (P < 0.05). (D) Changes in the mean SSC amplitude after bath application of NT-3. In each recording, the mean SSC amplitude was determined 20–25 min after NT-3 application and normalized to the mean SSC amplitude before NT-3 application.
	Figure 2 TrkC immunoreactivity is detected at different neuronal segments. (A–C) Representative examples of neurons stained with antibodies to TrkC (Santa Cruz Biotechnology). The immunofluorescence signal is evident at the soma (A), along the axon (A and B); and at the growth cone (C). (D) Control experiment demonstrating specificity of staining. Preincubation of primary antibodies with the blocking peptide largely abolished the immunofluorescence signal. (E–F) Neurons were stained with antibodies to TrkC (Upstate Biotechnology); this antibody was raised against the extracellular domain of TrkC receptor. The immunofluorescence signal can be detected at the cell body and along the axon (E) and at the distal axon (F).
	Figure 3 Local exposure of different axonal segments to NT-3 rapidly potentiates ACh secretion from the distant presynaptic nerve terminals. (A) Schematic representation of experimental approach. Neurons with an axon ≈300–400 μm in length and synaptic contact with a myocyte were chosen for experiments. Two glass micropipettes positioned in the vicinity of the neuron were used for local perfusion of a specific site with a culture medium containing 200 ng/ml NT-3. SSCs in the postsynaptic myocyte were recorded by using the whole-cell patch-clamp method. (B) Examples of current traces recorded from the postsynaptic myocytes. Arrow marks the onset of local perfusion of the neuron with the NT-3-containing culture medium. Gradual increase in the SSC frequency with time is evident in all three recording configurations. (C) Changes in the SSC frequency with time after the onset of local perfusion. Each data point represents the mean ± SEM of 5–12 experiments. No change in the SSC frequency was detected after perfusion of the cell body with the fresh culture medium (control) or with BDNF-containing culture medium (two lower lines). ∗, P < 0.05. (D) Mean SSC amplitudes for a period of 15–20 min after the onset of local NT-3-application normalized to the mean SSC amplitude for a 5-min period before NT-3 application.
	Figure 4 “Leakage” of NT-3 from the superfused region does not contribute to the potentiation of ACh secretion at the preformed synapses. (A) Membrane currents recorded from the myocyte in the preformed synapse. NT-3 (200 ng/ml) together with α-bungarotoxin (500 μg/ml) was locally applied to the middle axonal segment as in Fig. 3A. The start of local perfusion is marked by the arrow. The postsynaptic myocyte was ≈400 μm away from the site of drug application. Withdrawal of the pipette used for the removal of the superfused solution (arrow) resulted in the accumulation of α-bungarotoxin in the dish medium and the inhibition of SSC at the preformed synapse. (B) Membrane currents recorded from an innervated myocyte in the preformed synapse. Bath application of α-bungarotoxin (500 ng/ml, marked by arrow) resulted in the decrease in the frequency of SSCs. (C) Membrane currents recorded from the innervated myocyte before bath application of NT-3 application (5 ng/ml, marked by arrow) and for a period of 15–18 min after NT-3 application (right trace). (D) Quantitative analysis of the data. In each experiment, the mean SSC frequency for a period of 15–18 min after the drug application was normalized to the mean SSC frequency before drug application. Each data point represents the mean ± SEM of seven to nine experiments.
	Figure 5 Bath application of NT-3 potentiates neurotransmitter secretion at the synapses protected from direct exposure to NT-3. (A) Schematic representation of experimental approach. ACh secretion from the presynaptic nerve terminal was continuously monitored by whole-cell patch-clamp recordings from the innervated myocyte in spontaneously formed synapses. After establishing a baseline level of synaptic activity, NT-3 was applied to the bath (final concentration 50 ng/ml). The synaptic area was protected from exposure to NT-3 by continuous perfusion of the synaptic contact with a fresh culture medium through a glass micropipette. (B) Changes in the mean SSC frequency with time after the onset NT-3 treatment. Data points represent mean ± SEM of experiments in which NT-3 (filled circles, n = 13) or culture medium without NT-3 (open circles, n = 7) was added to the bath. ∗, P < 0.05. (C–E) Illustration of the degree of protection of the synaptic area from the acridine orange molecules applied to the bath. The pipette (P) was used for continuous perfusion of the synapse with a fresh culture medium (see A). Phase contrast (C) and fluorescent images before (D) and 5 min after (E) application of acridine orange (30 μM). Notice the bright staining of the myocyte (arrowhead) located ≈150 μm away from the protected region. No staining of the myocyte in the shielded region (arrows in C–E) was observed. Similar results were obtained in six different experiments.
	Figure 6 Protein synthesis and signaling to the soma are not required for the acute potentiation of ACh release induced by NT-3. (A) Pretreatment with anisomycin (40 μM) or with cycloheximide (10 μM) for 1 h before bath application of NT-3 does not prevent rapid potentiation of ACh release at spontaneously formed synapses. Changes in the SSC frequency with time after the onset of NT-3 treatment. Each data point represents the mean ± SEM of five experiments. ∗, P < 0.01. (B) Potentiation of ACh release at the distal axonal fragments. The axon was transected in the vicinity of the cell body (Insert) and the SSC frequency was measured. Bath application of NT-3 (marked by arrow) resulted in a characteristically rapid potentiation of ACh release from the distal axonal fragments innervating muscles. Each data point is a mean ± SEM of five experiments. ∗, P < 0.01.

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