QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 27 September 2005
doi: 10.1242/jcs.02603

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liou, J.-C. Articles by Kang, K.-H. Search for Related Content PubMed PubMed Citation Articles by Liou, J.-C. Articles by Kang, K.-H.

A rapid, nongenomic pathway facilitates the synaptic transmission induced by retinoic acid at the developing synapse

¹ Department of Biological Sciences, National Sun Yat-sen University, No. 70, Lein-Hai Rd., Kaohsiung City, 804, Taiwan
² Department of Pharmacology, College of Medicine, National Cheng Kung University, No.1, Ta-Hsueh Road, Tainan 701, Taiwan
³ Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, No.1, Ta-Hsueh Road, Tainan 701, Taiwan

View larger version (18K): [in a new window]   Fig. 1. Ca2+-dependent facilitation of spontaneous ACh secretion by RA. The continuous trace depicts the membrane currents 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 resulted from quantal ACh secretion. Bath application of RA at 30 µM dramatically enhanced spontaneous transmitter release, as seen by a marked increase in the frequency of spontaneous synaptic events. Samples of current events are shown below at higher time resolution. Scale bars are 1 nA, 20 seconds, and 1 nA, 50 milliseconds for the slow and fast traces, respectively. (B) Pretreatment of the culture with BAPTA-AM at 30 µM significantly abolished RA-induced facilitation of SSC frequency. (C) Summary of the effect of a Ca2+ chelator. Values are the mean and s.e.m. from 6-17 separate experiments. The SSC frequency from a single synapse was counted for a 6-minute period in control and a 6-minute period after RA application. The data were then averaged and normalized to control of the same synapse (n=6-17). The horizontal reference dotted line, which defines basal activity as `1' is shown for comparison. *P<0.05 compared with the control group (Student's t-test).

View larger version (23K): [in a new window]   Fig. 2. Extracellular Ca2+ is not involved in RA-induced synaptic facilitation. The culture medium was replaced with Ca2+-free Ringer's solution (A) or Cd2+ was added to block the Ca2+ channels (B), 1015 minutes prior to the experiment. Short arrows marks the application of RA. Scale bars: 1 nA, 20 seconds, and 1 nA, 50 milliseconds for slow and fast traces, respectively. (C) Summary of the effects of RA on SSC frequency in normal culture medium, Ca2+-free medium and in medium containing Cd2+ (n=8-17). *P<0.05 compared with the control group (Student's t-test).

View larger version (19K): [in a new window]   Fig. 3. Dependence of RA effect on Ca2+ mobilization from internal Ca2+ stores. Drugs that inhibit the Ins(1,4,5)P3 receptor [Xestospongin C (XeC); 2-aminoethoxydiphenylborate (2-APB)] or the ryanodine receptor [8-(dethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8); ruthenium red] were added to the cell cultures 2030 minutes before the experiment at final concentrations of: XeC, 2 µM; 2-APB, 50 µM; TMB-8, 3 µM; and ruthenium red, 10 µM. The effect of RA on SSC frequency was then evaluated in the presence of XeC (A), 2-APB (B), TMB-8 (C) and ruthenium red (D). Scale bars: 1 nA, 20 seconds. (E) Summary of the drugs effect. For comparison, the horizontal dashed line defines basal activity as `1'. The error bars refer to s.e.m. (n=517). *P<0.05 compared with the control group (Student's t-test).

View larger version (31K): [in a new window]   Fig. 4. Activation of PLC and PI 3-kinase are involved in RA-induced facilitation of SSC frequency. The continuous traces depict the effects of RA on frequency enhancement of SSCs in the presence of, a PLC inhibitor, U73122 (A, 5 µM), a G protein inhibitor, GDPßS (B, 5 mM), a PI3 kinase inhibitor, wortmannin (C, 100 nM) and LY294002 (D, 5 µM). Downward deflections are SSCs (Vh=–70 mV). Scale bars: 1 nA, 20 seconds.

View larger version (27K): [in a new window]   Fig. 5. Effects of various kinase inhibitors on RA-induced synaptic facilitation. The continuous traces A-F show the effect of RA on the SSC frequency in the presence of a PKC inhibitor, H-9 (100 µM), BIS (bisindolmaleimide, 10 µM), a board-spectrum tyrosine kinase inhibitor, genistein (100 µM), an inactive analog of genistein, daidzein (100 µM), a Src tyrosine kinase inhibitor, PP2 (10 µM) or a MAP kinase inhibitor, PD98059 (10 µM). Downward deflections are SSCs (Vh=–70 mV). Scale bars: 1 nA, 20 seconds.

View larger version (33K): [in a new window]   Fig. 6. Summary of the change of RA-induced SSC frequency facilitation in the presence of various drugs. Cultures were pretreated with various inhibitors and the effect of RA on SSC frequency were evaluated as indicated and in Fig 5. Bars indicate the s.e.m. (n=517). *P<0.05 compared with the RA treatment group (Student's t-test).

View larger version (25K): [in a new window]   Fig. 7. Effect of RA on the intracellular Ca2+ concentration. Xenopus nerve-muscle cultures were loaded with Fura-2 AM, and ratio fluorometric measurement of intracellular Ca2+ was made on a single motoneuron. Representative traces show F340/F380 ratio changes induced by perfusion the culture (at time 0) with RA in Ringer's solution (A), Ca2+-free Ringer's solution (B), or pretreatment with thapsigargin (TG, 2 µM) followed by perfusion with Ca2+-free Ringer's solution (C). (D) Summary of the RA-induced intracellular Ca2+ change ([Ca2+]i) in the presence of various inhibitors (U73122, 5 µM; LY294002, 5 µM; genistein, 100 µM, PP2, 10 µM). Representative traces are shown in the inset. Data are presented as mean ± s.e.m. (n=37). *P<0.05 compared with the control group (Student's t-test).