Article Figures & Data
Figures
- Fig. 1.
SSCs recorded from day 1 and day 3 natural synapses. The continuous trace depicts the membrane currents recorded from an innervated muscle cell in day 1 (a) or day 3 (b) Xenopusculture, using the whole-cell recording method (V H = −70 mV). The superimposed traces of 10 continuous events were shown below at higher time resolution. Calibrations: 200 pA, 40 sec, and 230 pA, 4 msec for the slow and fast traces, respectively. c, d, Histograms of the amplitude distribution of all SSC events observed froma and b, respectively.Arrows indicate the mean values. e, Composite graphs of SSC amplitude distribution for data obtained from seven synapses. The Cumulative Frequency refers to the proportion of the total events.
- Fig. 2.
Effect of myocyte contact on the spontaneous ACh release from nerve terminals in day 3 Xenopus cell cultures. a, Phase-contrast photograph shows that the motoneuron either may form natural synapses with myocytes (left) or stay alone (n, naive) (right). A myocyte was manipulated into contact with the nerve terminals of naive neuron (n) for the detection of ACh release. For natural synapses, the postsynaptic myocyte was mechanically destroyed and the scattered debris of the myocyte was then removed carefully by using a micropipette (long arrow), and another isolated neuron-free myocyte (m) was manipulated into contact with the “vacated” (v) nerve terminals. b, Spontaneous ACh release was recorded in either naive neuron (bottom trace) or vacated nerve terminals (top trace) by using an isolated myocyte manipulated into contact as a detector for the secretion (V H = −70 mV). Insetsrepresent superimposed traces of five continuous SSCs at higher time resolution. Note the smaller amplitude of SSCs at naive synapse compared with that at vacated synapse. Calibrations: 150 pA, 40 sec, and 150 pA, 5 msec for slow and fast traces, respectively.c, Histograms of the amplitude distribution for all SSC events observed from b. Arrows indicate the mean values. d, Cumulative frequency of amplitude distribution of all SSC events obtained from vacated (n = 10) and naive (n = 9) nerve terminals.
- Fig. 3.
Effect of NT-3 chronic treatment on the spontaneous ACh release of naive nerve terminals in day 3Xenopus cell cultures. a, One-day-old cultures were treated with NT-3 (2 nm), and a myoball was detached from culture substratum and moved to contact with the naive nerve terminals to build manipulated synapse at day 3. The myocyte was whole-cell voltage-clamped at −70 mV to record SSCs. Note an increase in the mean amplitude of SSCs at the synapse treated with NT-3 (bottom trace) compared with that at control (top trace). Calibration: 200 pA, 40 sec. b, c, SSC amplitude distribution of manipulated synapse at control or NT-3-treated naive nerve terminals. Arrows indicate the mean values. d, Cumulative frequency of amplitude distribution of all SSC events obtained from control (n = 5) and NT-3-treated (n = 8) neurons.
- Fig. 4.
Summary of the change of SSC amplitude and frequency at natural synapse. Day 1 cultures were treated with various kinds of drugs as indicated in the figure (NT-3, 2 nm; K252a, 1 μm; d-Tc, 50 μm; and NT-3 antibody, 1:200). The amplitude and frequency of SSCs of natural synapses were measured at day 3 after washout of the drugs. The error bars represent SEM, and the number of synapses tested in each group is indicated in parentheses. *, p < 0.05 as compared with control; #, p < 0.05 as compared with thed-Tc-treated group (Student’s ttest).
- Fig. 5.
Effect on evoked ACh release inXenopus cell cultures. a, Day 1 cultures were treated with various kinds of drugs as indicated in the figure (concentrations were the same as in Fig. 4), and ESCs of natural synapse were recorded with whole-cell voltage-clamped myocytes (V H = −70 mV) at day 3, after washout of the drugs. The presynaptic neuron was stimulated with an extracellular microelectrode at the soma to fire action potentials at a rate of 0.2 Hz. Oscilloscopic traces of five superimposed ESCs are shownbelow. Calibrations: 120 nA, 100 sec, and 1 nA, 10 msec, for the slow and fast traces, respectively. b,Bar graphs for quantitative comparison of ESC amplitudes. The numbers associated with the data refer to the total number of synapses examined. *, p < 0.05 as compared with control; #, p < 0.05 as compared with the d-Tc-treated group (Student’st test).
- Fig. 6.
Effect of d-Tc chronic treatment on the SSCs of natural synapse. Day 1 Xenopus cell cultures were treated with d-Tc (50 μm) and washed with Ringer’s solution at day 3 before the electrophysiological experiments. a, Continuous trace depicts membrane currents recorded from a whole-cell voltage-clamped myocyte (V H = −70 mV), which was innervated by control neuron (top) or chronically treated withd-Tc (bottom). Iontophoretic application of ACh at the myocyte surface was performed, as shown by the solid bar. Five continuous superimposed traces of SSCs or iontophoretic ACh-induced currents were shown below at higher time resolution. Calibrations: 200 pA, 20 sec, and 100 pA, 10 msec, for slow and fast traces, respectively. b, c, Histograms of SSC amplitude distribution of the same natural synapses as shown ina. Arrows indicate the mean values.d, Cumulative frequency of amplitude distribution of all SSC events obtained from control (n = 8) andd-Tc-treated (n = 7) synapses.
Tables
SSC amplitude (pA) Control NT-3 treatment Manipulated synapse Naive nerve terminal 50.9 ± 6.4 (9) 75.3 ± 5.8 (8)* Vacated nerve terminal 75.8 ± 7.4 (10) ND Natural synapse 155.3 ± 19.1 (27) 184.2 ± 33.1 (22) Natural synapse treated with d-Tc 48.9 ± 3.4 (12) 110.4 ± 13.2 (9)* -
Drugs were bath-applied to day 1 cultures and removed by several washes with Ringer’s solution on day 3 for the recordings of spontaneous synaptic currents (SSCs). SSCs were recorded from either manipulated synapses or natural synapses by whole-cell voltage-clamped myocyte (VH = −70 mV).
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The values were shown as mean ± SE (n). n, Number of synapses.
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ND, Not determined.
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↵* p < 0.05 as compared with control.
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