Wang T et al. (1995), Neurotrophins promote maturation of developing ...

XB-ART-19541

J Neurosci 1995 Jul 01;157 Pt 1:4796-805.

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Neurotrophins promote maturation of developing neuromuscular synapses.

Wang T , Xie K , Lu B .

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Although the effects of neurotrophins on survival and differentiation of various neuronal populations have been well studied, little is known about their role in synaptic development and function. We have investigated the long-term effects of neurotrophins in the maturation of neuromuscular synapses in Xenopus nerve-muscle cocultures. BDNF and NT-3, but not NGF, elicited significant changes in several properties of spontaneous synaptic currents (SSCs), indicative of more mature synapses. Most synapses treated by the neurotrophins exhibited a bell-shaped distribution of SSC amplitudes, which reflects mature quantal secretion. The neurotrophins also potentiated the efficacy and reliability of stimulus-induced synaptic transmission. Moreover, BDNF and NT-3 increased the levels of the synaptic vesicle proteins, synaptophysin, and synapsin 1 in the spinal neurons. The number of varicosities per neuron also showed a significant increase after neurotrophin treatment. The effects of the neurotrophins appear to be mediated by the Trk family of receptor tyrosine kinases, primarily through a presynaptic mechanism. These results suggest that BDNF and NT-3 promote functional maturation of synapses.

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Species referenced: Xenopus
Genes referenced: bdnf ngf ntf3 syn1
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	Figure 1. Effect of NT-3 on spontaneous synaptic activity in 2-d-old cultures. A, Membrane currents recorded from innervated myocytes from control (upper) or NT-3-treated (lower) cultures. Spontaneous synaptic currents (XSCs) of varying amplitudes are shown as downward deflections (V, = -70 mV, filtered at 150 Hz). Note the increase in frequency and mean amplitude of the SSCs at the NT-3-treated synapse. Calibration: 0.5 nA, 40 sec. B, Histograms of SSC amplitude distribution constructed from recordings of control and NT-3-treated synapses shown in A.
	Figure 2. Effect of NT-3 on spontaneous synaptic activity in 3-d-old cultures, A, Superimposed oscilloscopic traces of SSCs at higher time resolution (filtered at 2.5 kHz). Note the decrease in the rise time of SSCs after NT-3 treatment. Calibration: 0.5 nA, 1 msec. B, Composite amplitude distributions of SSCs. Three representatives from either control or NT-3-treated synapses are shown. The control data have been fitted by exponential curves, and the NT-3-treated data fitted by polynomial curves to show peaks of the bell-shaped distribution of SSC amplitude.
	Figure 3. Effect ‘of NT-3 on impulse-evoked synaptic activity. The continuous traces represent membrane currents recorded from innervated myocytes in 2-d-old cultures treated with (lower) and without (upper) NT-?. The presynaptic neuron was stimulated at the soma extracellularlv (0.5-5 V. 0.1 msec duration, 0.05 Hz) over the duration of the recording to initiate action potentials. Evoked synaptic currents (ESCs) appear as large, regularly spaced downward deflections marked by arrowheads in the current traces amidst randomly occurring SSCs (V,: = -70 mV, filtered at 150 Hz). Calibration: 2 nA, 0.5 min.
	Figure 4. Quantitative comparison of NT-3 effects on ESC amplitudes and variations in 2 d and 3 d cultures. Approximately 20 to 50 evoked currents were recorded from each individual synapse. The mean amplitudes and coefficient of variation (CV, defined as SD/mean of ESC amplitude) of ESCs were calculated individually for each group of synapses before averaging. Number of synapses used in each group: seven control and nine NT-3-treated synapses in 2 d cultures; and six control and seven NT-3-treated synapses in 3 d cultures. Error bars are standard errors. The data were subjected to Student’s t test. p < 0.05; *p < 0.01.
	Figure 5. Effects of NT-3 on synaptophysin expression. Dissociated, 3-d-old X~~opu.s nerve-muscle cultures were fixed and processed for standard indirect immunofluorescence histochemistry, using an anti-synaptophysin antibody. Note the increase of synaptophysin staining as well as the number of varicosities in BDNF- and NT-3-treated neurons. Scale bar, 20 pm.
	Figure 6. Effects of NT-3 on the expression of synapsin I. Three-day-old nerve-muscle cultures were stained with an antibody agamst Xenop~s synapsin I. Note the increase of synapsin I staining in BDNF- and NT-3-treated neurons. Also notice that the number of varicosities in cultures treated with BDNF and NT-3 were markedly increased. Scale bar, 20 pm.
	Figure 7. Quantitation of neurotrophin effects on the staining of synapsin I and synaptophysin. Images of spinal neurons in 3 d cultures grown in different conditions as indicated were analyzed by the Adobe PHOTOSHOP program. Data are expressed as percentage of control (mean -C SEM). Each column represents relative staining intensity on varicosities averaged from five to eight spinal neurons. *Significantly different (t test, p < 0.001).
	Figure 8. Scatted plot of neurotrophin effects on the number of KUicosities per neuron. Each data point represents number of varicosities of a single spinal neuron. The horizontal lines indicate the mean values of control and neurotrophin-treated groups. The BDNF and NT-3 groups are significantly different from the control group (t test, p < 0.001). The NT-3 + k252a group is not significantly different from the control group.