ON THE EXCITABILITY OF THE EPIDERMIS AND THE ATYPICAL EPIDERMIS OF CYNOPS ORIENTALIS EMBRYOS
Wu Wei-lin,Chen Yan-hua and Chuang Hiao-hui (Shanghai Institute of Cell Biology,Academia Sinica)
It has been reported that upon electricstimulus the atypical epidermis developed invitro from the isolated ectoderm of the earlygastrula did not display action potential(AP).If,however,the atypical epidermiswere grafted to a host embryo AP can be re-corded.During the course of further studiesit was observed that some of the atypicalepidermis do show excitability if they werestimulated with stronger stimulus.And,itwas also observed that the excitability of theepidermal cells varied if the experiments werecarried out in different seasons.Experimentswere carried out systematically to investigatethese phenomena. Atypical epidermis isolated from the ear-ly gastrula or from different developmentalstages and cultured in Holtfreter solution andthe epidermis of intact embryos were studiedwith the conventional intracellular micro-electrode recording technique.Atypical epider-mis were stimulated with electric stimulusat 6V at first,and,if they display AP thestrength was lowered to determine the thre-shold of AP.The duration of stimulus wasfixed at 1 mS. A.The Excitability of the Atypical Epi-dermis Isolated from Stage 10 (Early Gas-trula). The excitability of the in vitro cultiva-ted atypical epidermis was determined at dif-ferent stages corresponding to stages 26 to38.In all the stages AP appears in someof the specimens but the percentage varies.Instage 27 and stages 36—37 the percentageis higher,and in stages 28—34 AP appearsonly in a few specimens (Tab.1).In thisrespect the atypical epidermis is differentfrom the epidermis of the intact embryoswhich displays AP in all individualsbetween stages 26 and 37 after electric sti-mulation. The average threshold of AP of the aty-pical epidermis is obviously higher than thatof the epidermis of intact embryos in corres-ponding stages (p0.001).On the otherband,in both the epidermis of the intactembryos and the atypical epidermis the avera-ge threshold of AP was higher at the timewhen the AP first appears and before its dis-appearance (Tab.1 and 3) The distribution of the threshold of APamong the explants determined at the samedevelopmental stage was more scattered than that of the epidermis of intact embryos.Forexample,when determined at stage 27 therange was 3.3—6.0 V,and that of the epi-dermis of intact embryo was 1.7—2.3 V.When determined at stage 36,the correspon-ding ranges were 1.9—6.0 V and 1.4—2.2V respectively (as determined in May andJune). Since in the above experiments the sti-mulating and the recording electrodes wereplaced at a distance to each other,in orderto acertain whether the absence of AP insome of the explants was caused by the non-conductivity or the deficiency of excitabilityof the epidermal cells,experiments with asingle electrode both for stimulation and re-cording were carried out with atypical epi-dermis isolated from stage 10.When testedat the time corresponding to stage 27 (n=18);stage 32 (n=36),stage 35 (n=15),stage 36(n=23) and stage 38 (n=15),16.7%,26.1%,5.5%,13.5% and 20.0% of theexplants displayed AP.These results are inagreement with those stated above,and it isthus made clear that the absence of AP inatypical epidermis is caused by the deficiency of excitability but not the non-conductivityof the cells. The excitability of the cells within anatypical epidermis did not develope uniformly.It was observed that with the same strengthof stimulation only some cells of an atypi-cal epidermis displayed AP. B.The Excitability of the Atypical Epi-dermis Isolated from Different Stages. Ectoderm from embryos at stages 4,6,7,10,11,13 or ventral epidermis fromstages 15 and 17 were isolated and culturedin vitro and their excitability was determinedat different developmental stages i.e.stages27.33—34.36—37.It can be seen fromTab.2 that,like the results stated above,inall of the experimental series,no matter theatypical epidermis was isolated from whichstage,the percentage of specimens displayedAP was obviously higher when determined instage 27 and in stages 36—37.And,as Tab.1 and 3 show,the average threshold of APof atypical epidermis isolated from stages 1013.15 and 17 is higher when the AP firstappears and at the time before its disappea-rance. Furthermore,from Tab.2 the influenceof the age of donor embryo on the exci-tability of the atypical epidermis can also beseen.The percentage of specimens showed APis lower in the series isolated at early stagesand higher in those isolated at later stages,no matter the determination was performed atstages 27,33—34 or 36—37.It is obviousthat the neighbouring tissues in the donor em-bryos have already exerted some influence onthe epidermis at the time of isolation. From the above stated both in A.and B.it can be concluded that the atypical epider-mis in general is probably excitable.But theexcitability in general is so weak,that theydo not display AP if the stimuli used werenot strong enough.It was also observed thatamong those explants which were non-exci-table at 6 V,some of them may still displayAP if the strength of stimulus was raised to8 V.The fact that the atypical epidermis wasisolated at early stages of development and cultured in vitro,the excitability of the aty-pical epidermal cells should be considered asan independent functional differentiation ofthese cells,the excitability occurs when thecells developed to a certain state of differen-tiation. C.Seasonal Changes of the Thresholdof Excitability of Epidermal Cells. The natural breeding season of Cynopsorientalis is from March to May.Under ourlaboratory condition the breeding season canbe prolonged from March to the beginning ofAugust.The threshold of AP of epidermialcells varied within this period.This pheno-menon was examined with the in situ epider-mal cells at first.From Tab.4 it can be seenthat when the intact embryos were examinedat stages 26,27,29,32 or 37,the averagethreshold of AP was higher in May—Junethan in March,and the difference betweenAugust and May—June was even more con-spicious. The same experiments were also carriedout with the atypical epidermis isolated fromstage 10 embryos.The average threshold ofAP of stages 27,33,36,37 and 38 wassignificantly higher when measured in May—June than those measured in March. Since there exists a seasonal change inthe excitability of the cells,it seems necessaryto notify the time when the experiments werecarried out when describing the experimentalresults.