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First published online 25 May 2004
doi: 10.1242/jcs.01153

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Non-genomic regulation of transmitter release by retinoic acid at developing motoneurons in Xenopus cell culture

Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung City 804, Taiwan

View larger version (22K): [in a new window]   Fig. 1. Effect of retinoic acid (RA) on the spontaneous ACh quantal release at Xenopus neuromuscular synapses. (A) The continuous trace depicts the membrane current recorded from an innervated myocyte in day-1 Xenopus cell culture, using the whole-cell recording method (VH= -70 mV, filtered at 10 kHz). Downward events are inward currents resulting from quantal ACh secretion. Samples of current events are shown below at higher time resolution. Scale bars are 1 nA, 20 seconds for the slow traces, and 1 nA, 50 milliseconds for the fast traces. (B) The presynaptic neuron was stimulated to fire action potentials and ESCs recorded in the myocytes are shown as downward deflections at the times marked by arrowheads. Shown below at higher time resolution are ESCs before and after RA application. Scale bars are 1 nA, 20 seconds, and 1 nA, 20 milliseconds for the slow and fast traces, respectively. (C) Amplitude distribution of all SSC events before and after RA treatment. Events during a period 10-15 minutes after the application of RA were analyzed. The cumulative frequency refers to the proportion of total events with amplitudes smaller than the given amplitude. Each value represents mean±s.e.m. from 10 experiments. There was no significant difference between the two distributions (P>0.05, Kolmogorov-Smirnov test). (D) Concentration-response relationship for RA on the potentiation of SSC frequency. The mean SSC frequency 10-15 minutes after the application of RA at different concentrations was normalized for each synapse by setting the mean SSC frequency before the RA addition to 1. Each value represents the mean and vertical line representing the s.e.m. for 5-17 experiments.

View larger version (24K): [in a new window]   Fig. 2. RA itself is responsible for the potentiation of SSC frequency. RA was decomposed into 5,6-epoxyretinoic acid and glucuronide by exposing 10 µM RA solution to a strong light overnight and the complete decomposition was confirmed by disappearance of yellow color of the RA solution ('bleached RA'). The basal level of spontaneous ACh release was not affected by the addition of bleached RA (A), DMSO vehicle (B), or (C) retinol. Note the subsequent addition of RA 20 minutes after retinol application caused a significant increase in SSC frequency. Scale bars, 1 nA, 30 seconds. (D) Summary of the change in SSC frequency. Plotted are the mean and s.e.m. from 7-9 separate experiments. *P< 0.05 compared with the control group (Student's t-test). The reference dotted line is shown for comparison.

View larger version (19K): [in a new window]   Fig. 3. SSC frequency facilitation induced by RA requires continued presence of the factor. (A) The continuous trace depicts the inward membrane current recorded from an innervated muscle cell in Xenopus culture (filtered at 10 kHz). The arrow marks the time of addition of RA to the culture medium and RA was removed by the substitution of the culture medium with Ringer's solution at the time indicated by `wash'. Scale bars, 2 nA, 20 seconds and 1 nA, 50 milliseconds. (B) Changes in the SSC frequency (normalized to control frequency) with time during the addition and removal of 30 µM RA. The increased SSC frequency declined to control level within 5 minutes of washout of RA.

View larger version (31K): [in a new window]   Fig. 4. Protein synthesis is not required for SSC frequency facilitation induced by RA. The culture was pretreated with 30 µM anisomycin (A) or 30 µM cycloheximide (B) for more than 45 minutes and the effect of RA on SSC frequency was then evaluated. Scale bars, 500 pA, 20 seconds. (C) Summary of data for the SSC frequency (±s.e.m.) at 10-15 minutes after RA application under various drug pretreatment, normalized by mean control values from the same synapse before the addition of RA.

View larger version (27K): [in a new window]   Fig. 5. Effect of various subtype-specific synthetic compounds on the SSCs in Xenopus cell cultures. The continuous traces depict the membrane currents recorded from the innervated myocytes before and after bath application of various agonists (10 µM). Downward deflections are SSCs (VH= -70 mV). Scale bars, 1 nA, 20 seconds.

View larger version (21K): [in a new window]   Fig. 6. Summary of the change in SSC frequency resulting from addition of various retinoid derivatives. Bar graph represents mean increase in SSC frequency induced by a series of retinoid derivatives; the name for each is indicated below the bars. Each bar shows the mean (±s.e.m.) compared to control from 7-9 cell cultures. For comparison, the horizontal dashed line defining basal activity as 1 is shown. *P<0.05 compared to the RA treatment group (Student's t-test).