Sodium and Calcium Currents in Dispersed Mammalian Septal Neurons

Sodium and Calcium Cu en s in Dispe sed Mammalian Sep al Neu ons ANTONIO CASTELLANO and Jos~ L6PEZ-BARNEO F om he Depa amen o de Fisiolog a y Bio lsica, Facul ad de Medicina, 41009 Se illa, Spain A B S T R A C T Vol age-ga ed Na ÷ and Ca 2÷ conduc ances o eshly dissocia ed sep al neu ons we e s udied in he whole-cell con igu a ion o he pa ch-clamp echnique. All cells exhibi ed a la ge Na + cu en wi h cha ac e is ic as ac i a ion and inac i a ion ime cou ses. Hal - ime o peak cu en a -20 mV was 0.44 _ 0.18 ms and maximal ac i a ion o Na + conduc ance occu ed a 0 mV o mo e posi i e memb ane po en ials. The a e age alue was 91 -+ 32 nS (~ 11 mS cm-2). A all memb ane ol ages inac i a ion was well i ed by a single exponen ial ha had a ime cons an o 0.44 _+ 0.09 ms a 0 inV. Reco e y om inac i a ion was comple e in ~900 ms a -80 mV bu in only 50 ms a -120 mV. The decay o Na ÷ ail cu en s had a single ime cons an ha a -80 mV was as e han 100 p.s. Depola iza ion o sep al neu ons also elici ed a Ca ~+ cu en ha peaked in ~ 6-8 ms. Maximal peak Ca ~÷ cu en was ob ained a 20 mV, and wi h 10 mM ex e nal Ca ~+ he ampli ude was 0.35 - 0.22 hA. Du ing a main ained depola iza ion his cu en pa ially inac i a ed in he cou se o 200-300 ms. The Ca ~+ cu en was due o he ac i i y o wo ypes o conduc ances wi h di e en deac i a ion kine ics. A -80 mV he closing ime cons an s o slow (SD) and as (FD) deac i a ing channels we e, espec i ely, 1.99 -+ 0.2 and 0.11 +_ 0.03 ms (25°C). The wo kinds o channels also di e ed in hei ac i a ion ol age, inac i a ion ime cou se, slope o he conduc ance- ol age cu e, and esis ance o in acellula dialysis. The p opo ion o SD and FD channels a ied om cell o cell, which may explain he di e en ial elec ophysiological esponses o in acellula ly eco ded sep al neu ons. INTRODUCTION Mammalian cen al neu ons possess in insic elec ical p ope ies ha make hem able o gene a e complex i ing pa e ns. This implies he exis ence o a a ie y o memb ane ionic conduc ances which, wi h a ew excep ions, a e poo ly cha ac e ized. Some p ope ies o Na + channels ha e ecen ly been s udied in dissocia ed hippo- campal (Sah e al., 1988) and neoco ical (Huguena d e al., 1988) neu ons, as well as in Pu kinje cells o o gano ypic ce ebella cul u es (G~ihwile and Llano, 1989), bu addi ional wo k is s ill needed o ully in es iga e he biophysical p ope ies o ce eb al Na ÷ channels and o asce ain whe he hei cha ac e is ics a e homoge- neous in di e en b ain egions. Ca ~+ channels, on he o he hand, ha e been Add ess ep in eques s o D . J. L6pez-Bameo, Depa amen o de Fisiologia M dica y Bio sica, Facuhad de Medicina, A da. S~inchez Pizju in, 4, 41009 Se illa, Spain. J. GEN. PHYSIOL. © The Rocke elle Uni e si y P ess • 0022-1295/91/02/0303/18 $2.00 Volume 97 Feb ua y 1991 303-320 303 on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 304 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 . 1991 in ensi ely s udied in neu ons o he pe iphe al ne ous sys em (Fedulo a e al., 1985; Ca bone and Lux, 1987; Fox e al., 1987; Swandulla and A ms ong, 1988), bu wi hin he b ain hey ha e been only pa ially cha ac e ized (He u~indez-C uz and Pape, 1989; Meye s and Ba ke , 1989). We ha e p e iously shown ha in acel- lula ly eco ded sep al neu ons in in i o b ain slices display di e en ial elec ical esponses due o he a iabili y in he dis ibu ion o memb ane ionic conduc ances (L6pez-Ba neo e al., 1985; Al a ez de Toledo and L6pez-Bameo, 1988). This a icle con inues ou p eceding wo k and ocuses on he cha ac e iza ion o he ionic cu en s o eshly dissocia ed sep al neu ons om adul guinea pigs. Due o hei size and egula geome y hese cells p o ide an excellen p epa a ion o s udying he ol age-ac i a ed ionic channels in a well-clamped neu onal memb ane using he whole-ceU a ian o he pa ch-clamp echnique (Hamill e al., 1981). This esea ch no only has biophysical in e es , i is also o impo ance because an accu a e analysis TABLE I Composi ien o Solu ions NaCl KC! CaClz MgCi~ BaCI~ CoCI 2 T izma* HEPES Ex~ nal 130 Na 130 2.7 2.5 1 -- -- -- 10 80 Na 80 2.7 2.5 1 -- -- 70 -- I0 Ca 70 2.7 10 -- -- -- 60 -- 10 Ba 70 2.7 -- -- I0 -- 60 -- Mg/Co 70 2.7 1 7.5 -- 1.5 60 -- NaCI KCI K-glu ama~ KF MgCI, CsCI CsF HEPES EGTA In~ nal 130 K -- 30 80 20 2 -- -- 10 5 130 Cs .... 2 110 20 10 5 40 Na 40 -- -- -- 2 70 20 10 5 All alues a e gi en in miUimola . pH was 7.4 in ex e nal and 7.3 o 7.35 in in e nal solu ions *T is (hyd oxyme hyl)aminome hane (pH = 7.4). o he ionic conduc ances o cen al neu ons is equi ed be o e unde aking he in es iga ion o hei modula ion by neu omodula o s and d ugs. This a icle concen a es on he desc ip ion o he p ope ies o Na ÷ and Ca ~÷ cu en s; he K ÷ cu en is only b ie ly p esen ed and he e o e no s udied in de ail. METHODS Cells we e ob ained om co onal slices, 300-400 p~m hick, o he adul guinea pig sep al egion. The p ocedu e ollowed o slicing was he same as p e iously desc ibed (Al a ez de Toledo and L6pez-Bameo, 1988). Fo cell dissocia ion, six slices con aining only he sep al nucleus we e placed in a ial wi h 3 ml o Ea le solu ion (Flow Labo a o ies, Inc., McLean, VA) con aining 0.2 mg/ml o ypsin ype IX and 0.05 mg/ml o DNase ype II (Sigma Chemical Co., S . Louis, MO). This solu ion was con inuously bubbled wi h a mix u e o 95% O~ and 5% CO2 and he issue was incuba ed o 20 min a 37°C. A e his ime pe iod he issue was washed on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 CASTELLANO AND I.XSPEZ-BARNEO Na + and Ca 2+ Channels o Sep al Neu ons 305 wice and he enzymes we e emo ed. Cells we e mechanically dispe sed using i e-polished Pas eu pipe es, cen i uged, and esuspended in MEM cul u e medium supplemen ed wi h glu amine, an ibio ics, and bo ine e al se um. Cells we e pla ed on agmen s o glass co e slips ea ed wi h poly-L-lysine and main ained in an incuba o un il use (be ween 2 and 6 h a e pla ing). The gene al p ocedu es and he se up used o eco ding o whole-cell cu en s we e he same as p e iously desc ibed (U e ia e al., 1989a). The composi ion o he eco ding solu ions is gi en in Table I. Juncdon po en ials we e co ec ed once he elec ode was in he ba h and immedia ely be o e es ablishing a seal. In he ex and in he igu e legends solu ions a e indica ed as ex e nal//in e nal. Expe imen s we e pe o med a oom empe a u e (20-25°C). Fo eco ding we used a pa ch-clamp ampli ie buil in ou labo a o y ollowing he design o Sigwo h (1983). A as and uni o m con ol o he memb ane po en ial was a o ed by he use o low esis ance elec odes (be ween 0.8 and 2 M ~), a eedback esis ance o only 100 Ml'l in he cu en - o- ol age con e e , and elec onic compensa ion o se ies esis ance. Wi h his eco ding con igu a ion capaci y ansien s ypically had ime cons an alues be ween 40 and 50 ~s. Clamp speed was u he enhanced by ballis ic cha ging o memb ane capaci ance (Ma eson and A ms ong, 1984). Compensa ion o se ies esis ance was done empi ically; in mos expe imen s we compensa ed o 1.5 imes elec ode esis ance. An IBM-PC/AT compu e in e aced o he analog elec onics was used o pulse gene a ion and o acquisi ion, display, s o age, and analysis o he da a. The in e ace and he associa ed so wa e we e de eloped and ab ica ed in ou labo a o y (U e ia e al., 1989b) and allowed us sub ac ion o he linea ionic and capaci y cu en s. Sampling in e al was ei he 10 o 20 Ws. Tail cu en s we e i ed wi h one o he sum o wo exponen ials using a leas -squa es p ocedu e. RESULTS Cells subjec ed o ol age-clamp had an almos sphe ical shape and no app eciable p ocesses unde he ligh mic oscope. The diame e o he dispe sed neu ons a ied be ween 12 and 25 Ixm. Memb ane capaci ance, calcula ed om he ime in eg al o he ansien capaci y cu en s, was 8 - 3 pF (mean _ SD, n = 87). Inpu esis ance was 1.5 -+ 0.6 G /. Whole-Cdl Cu en s Fig. 1 A is a ep esen a i e example o he majo cu en componen s eco ded in sep al neu ons du ing a s ep depola iza ion o 20 mV. A he beginning o he pulse he cell gene a es a as inwa d cu en ha peaks in less han 0.5 ms. In he cou se o he nex 2 ms he cu en changes om inwa d o ou wa d and ises slowly un il he end o he pulse. On e u n o he holding po en ial he e is an almos ins an aneous jump in he cu en (which in his eco ding las s less han 80 V,s) and a la ge ail cu en is obse ed. Fig. 1 B shows he cu en eco ded a e blockade o K + channels by in e nal Cs +. The ou wa d cu en is supp essed, and a e he ini ial as inwa d componen a second, mo e s eady componen is disclosed, which, when he pulse is u ned o , is ollowed by he ail cu en . Ex e nal addi ion o e odo oxin (TTX, 0.5-1 ~M) abolishes he as inwa d cu en , while he s eady componen and he ail emain unal e ed (Fig. 1 C). I will be shown la e ha bo h he slow inwa d cu en and he ail disappea a e emo al o ex e nal Ca 2+. These indings indica e ha he di e en componen s o he whole-cell cu en a e due o he ac i i y o Na ÷, Ca 2÷, and K ÷ channels. The h ee cu en componen s we e eco ded in e e y neu on on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 306 : / "-: / ./ u THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 • 1991 B #. 2 ms C ,.-.. FIGURE 1. Majo cu en compo- nen s in sep al neu ons. The h ee cu en sweeps we e eco ded du ing 6-ms ol age s eps o 20 mV om a holding po en ial o -80 mV. Solu- ions: 130 Na//130 K (A), 10 Ca//130 Cs (B and C). In C, 1 IxM TIXwas added o he ex e nal solu ion. s udied (n > 150), al hough he ampli ude o each a ied om cell o cell. In he ollowing sec ions we analyze he p ope ies o Na + and Ca *+ cu en s. Na + Channels Ac i a ion and conduc ance- ol age ela ions. Na + cu en was s udied in isola ion in cells dialyzed wi h he 130 mM Cs +'solu ion and ba hed in an ex e nal solu ion wi h 130 mM Na +, whe e 90% o he Ca ~+ was eplaced by Mg ~+ and Co s+ (see Table I). In hese solu ions depola iza ion elici ed la ge Na + cu en s ha a 0 mV had a peak ampli ude o 4.6 + 1.6 nA (n = 21). Assuming no memb ane in olding, cu en densi y was 0.6 mAcm -~. Mos expe imen s we e, howe e , pe o med wi h he 80 Na ex e nal solu ion (Table I) o a o an accu a e con ol o he memb ane po en ial. Fig. 2 illus a es a cu en ace ob ained in low ex e nal Na +. The eco ding is e y ep esen a i e o a well-con olled Na + cu en wi h as ac i a ion 80 Na. TTX : ' l • - -80 Na • :" 1ms • i FIGURE 2. Na + cu en eco ded on isola ion du ing a ol age pulse om -80 o 0 mV. The a ows indica e he onse and he e mina ion o he pulse. Solu ions we e 80 Nail130 Cs. The cu en was comple ely sup- p essed by 1 p,M TFX. on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 CASTELLANO AND LOPEZ-BARNEO Na + and Ca 2* Channels o Sep al Neu ons 307 and comple e inac i a ion in 5-6 ms. The igu e also shows ha addi ion o TTX p oduced a comple e blockade o he cu en . Wi h 80 mM ex e nal Na ÷, peak cu en ampli ude a 0 mVwas 2.8 - 1.3 nA (n = 49). Good con ol o he memb ane po en ial was also indica ed by a conduc ance- ol age cu e wi h a sigmoid ising phase (no shown), and because cu en s o di e en ampli ude eco ded a he same memb ane po en ial (as, o ins ance, hose in Fig. 6A) had, a e scaling, almos iden ical ime cou ses. Fig. 3 A is a amily o Na ÷ cu en s gene a ed by depola iza ion o he indica ed memb ane po en ials. In his expe imen we added 40 mM Na + o he in e nal solu ion o ob ain a well-de ined e e sal po en ial (EN=). Cu en is inwa d a /.~ • ~ -50 o +90 :"J / "" l ." / :), 2 nA ~j 2 ms B I M.nA -40 -20 20 /40 _. I I I ._[ I -0.7 -1.4 -1.4 / -0.7 6O I VM,mV FIGURE 3. Na + I-V ela ions. (A) Family o cu en sweeps elici ed by depola iza ions o -50, -30, -10, 10, 30, 50, 70, and 90 mV om a holding po- en ial o -80 inV. (B) Peak cu en ampli ude (o dina e) a a ious memb ane po en ials (abscissa). In e sec ion o he con inuous line wi h he ab- scissa gi es he alue o he e- e sal po en ial (31 mV). Solu- ions: 130 Na//40 Na. memb ane po en ials mo e nega i e han EN, and ou wa d a mo e posi i e po en- ials. The cu en u ns on apidly and inac i a es comple ely a all ol ages. Ac i a ion kine ics speed up wi h memb ane depola iza ion and hal - ime o peak cu en is 0.44 + 0.18 ms a -20 mV bu only 0.21 --. 0.06 ms a +20 mV (n = 10). Fig. 3 B is he I-V cu e ob ained by measu ing he peak cu en a a ious memb ane po en ials. This igu e shows ha a measu able cu en can be eco ded a -50 mV and ha he maximal peak cu en is ob ained be ween -20 and -10 mV. The e e sal po en ial o he cu en occu s a +31 mV, which is simila o he Na ÷ equilib ium po en ial calcula ed by he Ne ns equa ion. This sugges s ha , as in o he p epa a ions (Fenwick e al., 1982; Ma eson and A ms ong, 1984; U e ia e al., 1989a), he aqueous componen s o he cy osol equilib a e comple ely wi h he on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 308 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 97 • 1991 pipe e solu ion. Na + conduc ance (gN:), calcula ed by he o mula gN: ---- IN:~ (I'M- ENd) (Hodgkin and Huxley, 1952), was maximal a 0 mV o mo e posi i e ol ages. The a e age alue om ou sep al neu ons was 91 - 32 nS (~ 11 mS cm-2), and hal o he o al conduc ance was ac i a ed a -35 mV. Inac i a ion. Du ing a main ained depola iza ion he inac i a ion o Na + cu - en s could be well i ed by a single exponen ial unc ion. This is illus a ed by he eco ding o Fig. 4 A. In he ange be ween -20 and +60 mV inac i a ion ime cons an diminishes wi h memb ane depola iza ion (Fig. 4 B) and a 0 mV i has an a e age alue o 0.44 --+ 0.09 ms (n = 5). The ol age dependence o s eady-s a e inac i a ion is illus a ed in Fig. 5. In A i is B 0.6- E ~0,4- .~ 0,2- A 1ms 4._ i i i i i -40 -20 0 210 40 60 V M,m FIGURE 4. Time cou se o Na + cu en inac i a ion. (A) Single sweep o a Na ÷ cu en eco ded du ing a depola iza- ion o 0 inV. A single exponen- ial ex apola ed o he onse o he pulse was i ed o inac i a- ion ime cou se. To acili a e compa ison, he exponen ial is d awn supe imposing he cu - en ace. (B) Dependence o inac i a ion ime cons an on he pulse memb ane po en ial. Ci cles a e he mean and he ba s he s anda d de ia ion o alues om i e cells. Solu ions: Mg/Co//130 Cs. shown ha he ampli ude o he cu en eco ded by a es depola iza ion o 0 mV depends on he memb ane po en ial du ing a condi ioning p epulse las ing 50 ms. Fig. 5 B shows he ela ion be ween he noninac i a ed ac ion o channels (o dina e) and he p epulse memb ane po en ial (abscissa) wi h mean alues ob ained om six cells. A -80 mV only a small (5-10%) ac ion o he channels a e inac i a ed, bu all o hem a e inac i a ed a ol ages mo e posi i e han -20 inV. The a e age hal s eady-s a e inac i a ion occu s a a memb ane po en ial o abou -50 mV; his pa ame e a ied be ween -45 and -70 mV in he ceils s udied. Reco e y om inac i a ion was s udied by applying a 20-ms condi ioning pulse o 0 on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 CASTELLANO AND L6PEZ-BARNEO Na + and Ca 2+ Channels o Sep al Neu ons 309 mV o inac i a e he Na ÷ cu en and, a e a a iable ime in e al a he holding po en ial, applying a es pulse o open he channels al eady deinac i a ed. The aces o Fig. 6 A a e Na ÷ cu en s elici ed by depola iza ion o 0 mV in he absence o p epulse (con ol) and 10, 25, and 900 ms a e he p epulse; inac i a ion is almos comple ely emo ed in he cou se o 900 ms. In Fig. 6 B a e shown he ime cou ses o eco e y om inac i a ion when he memb ane po en ial du ing he in e al is held a -80 (ci cles), -100 (squa es), o -120 mV ( iangles). Reco e y is abou six imes as e a -120 mV han a -80 inV. In e es ingly, a -80 mV he eco e y o he las 15-20% o he cu en equi ed as much as 900 ms. This indica es he p esence o a as inac i a ing componen ha eco e s ex emely slowly. Closing kine ics. Closing kine ics o mammalian neu onal Na + channels ha e no been p e iously s udied. We o en ob ained eco dings whe e he as Na ÷ ail A -20 / • '" 1hA 1 ms B 1,0- <E0.5- -100 -60 -20 M , mV FIGURE 5. S eady-s a e inac i a ion o Na ÷ cu en s. (A) Cu en sweeps du ing depola iza ions o 0 mV p e- ceded by a 50-ms p epulse o he indica ed memb ane po en ials. Holding po en ial, -80 InV. No mal- ized ac ion o peak cu en eco ded du ing he pulse (o dina e) as a unc- ion o he p epulse memb ane po- en ial. Ci cles and ba s a e he mean and s anda d de ia ion alues om i e cells. The inse illus a es he pulse p o ocol. Solu ions: Mg/Co//130 Cs. cu en s could be esol ed. The p o ocol consis ed o he applica ion o a b ie depola iza ion (0.3-0.5 ms) o open Na ÷ channels ha ended a he ime when he cu en was maximal (Fig. 7 A ). A he ins an o epola iza ion he d i ing o ce o Na ÷ suddenly inc eases and hus a ail cu en is eco ded. The decay o he ail e lec s he p og essi e deac i a ion o he channels open du ing he pulse. In ou expe imen s Na ÷ ail cu en s we e always well i ed by a single exponen ial unc ion (Fig. 7 B), which a -80 mV had a ime cons an as e han 100 Izs (n = 5). on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 310 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 . 1991 A /10 ms ~~ lnA con ol ~ ms >. 0.5 i i I -// I i 25 50 75 100 250 9()0 ime. ms FIGURE 6. Reco e y om in- ac i a ion o Na ÷ channels. (el) Na + cu en s elici ed by ol age s eps o 0 mV a he indica ed ime in e als a e a p epulse o 0 mV las ing 20 ms. The con ol ace was ob ained in he absence o p epulse. Hold- ing po en ial, -80 mV. (B) No malized pulse cu en am- pli ude (o dina e) as a unc ion o in e al du a ion (abscissa). Memb ane po en ials du ing he p epulse we e -80 mV (ci - cles), -100 mV (squa es), and -120 mV ( iangles). Lines a e i ed by eye. Solu ions: Mg/Co// 130 Cs. Ca 2+ Channels Iden i ica ion and I-V ela ions. In he p esence o TTX depola iza ion elici ed in all neu ons a slowly inac i a ing inwa d cu en ha was eadily iden i ied as a Ca ~+ cu en . Fig. 8 A is a ypical eco ding o he slow inwa d cu en . This ace shows ha he cu en u ns on slowly and almos eaches a maximum by he end o he 6-ms pulse. On epola iza ion he e is a ail cu en due o he inc ease in he elec ochemical g adien o Ca 2÷ ions. As in mos p epa a ions s udied, he cu en was la ge when Ba 2+ ions ins ead o Ca 2÷ we e used as cha ge ca ie (Fig. 8 B). Cu en du ing he pulse and a he pulse end ( ail) was supp essed a e emo al o A • ,. / loA :: 0,5 ms gle exponen ial wi h a ime cons an o 60 p~s. The exponen ial epola iza ion. Solu ions: Mg/Co//130 Cs. FIGURE 7. Closing kine ics o ce eb al Na ÷ channels. (A) Na + cu en eco ded du ing a 0.5-ms ol age pulse (indica ed be ween he a ows) o 0 mV om a holding po en ial o -80 inV. When he pulse is u ned o a as ail cu en is eco ded. (B) Fi ing o he de- ac i a ion ime cou se by a sin- is ex apola ed o he ins an o on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 CASTEL .~O AND L6PEz-B~Eo Na + and Ca z+ Channels o Sep al Neu ons 311 ex e nal Ca ~+ o a mix u e o Mg 2÷ and Co ~+, ca ions p ac ically impe mean h ough Ca ~+ channels (Fig. 8 C). Fig. 9 shows a amily o Ca ~÷ cu en s eco ded on depola iza ion o he indica ed memb ane ol ages. Cu en du ing he pulse, which is indica ed be ween he a ows, inc eases in ampli ude and ac i a es mo e apidly a mo e depola ized le els. A e a maximum he pulse cu en diminishes wi h posi i e po en ials due o he dec ease in he elec ochemical g adien o Ca 2+. Ca ~+ cu en measu ed a he end o he 6-ms s eps a a ious memb ane po en ials is illus a ed in Fig. 10. This/-V cu e shows ha he cu en becomes de ec able a -40 o -50 mV, eaches a maximum a abou +20 mV, and a e wa ds declines as he d i ing o ce o Ca ~+ p og essi ely dec eases. In 10 mM ex e nal Ca 2+ he ampli ude o he cu en measu ed a he end o 6-ms pulses o 20 mVwas 0.35 -+ 0.22 nA (n = 12). Sepa a ion o wo Ca 2÷ channel ypes. I is clea ly seen in Fig. 9 ha a e small depola iza ions he decay o he ails ollows a simple, slow ime cou se, bu a e depola iza ions o po en ials mo e posi i e han 0 mV a la ge as componen also A 10 Ca ";~"~.,,4.~_~. " ..... 1,-,;-.~-~""- FIGURE 8. Iden i ica ion o Ca 2+ cu - -~'''-~ .¢ en s. (A) Cu en p oduced by depo- ' la iza ion o 10 mV du ing 6 ms wi h Ca ~÷ as cha ge ca ie . (B) Cu en B eco ded in he same cell and wi h he 10 Ba .-, same pulse, using Ba ~+ ins ead o . ,,~ Ca 2÷ in he ex e nal solu ion. (C) : Blockade o he cu en a e eplace- : men o Ca 2+ by Mg ~+ and Co ~÷. : 1 nA Holding po en ial, -80 mV. The di - : 2 ms e en ex e nal solu ions a e indica ed C in he igu e; he in e nal solu ion was Mg. Co 130 Cs. ~"*"~'~"~'~"~'~'--'~.2" " appea s. Bo h he slow and as componen s o he ails g ow la ge as he memb ane po en ial is made mo e posi i e. The sudden appea ance o he as componen o he ails a ol ages be ween 0 and 20 mV pa allels he sha p ansi ion seen in he I-V cu e a he same ol ages (Fig. 10). These obse a ions sugges he exis ence in sep al neu ons o a leas wo di e en Ca 2÷ conduc ances wi h di e en ac i a ion ol ages and deac i a ion kine ics. These conduc ances a e p obably due o he ac i i y o wo kinds o Ca ~÷ channels. Sepa a ion o he wo componen s o he Ca ~+ cu en was done ollowing he me hod illus a ed in Fig. 11 (Co a, 1986; Ma eson and A ms ong, 1986; Taba es e al., 1989). T ace A is a ail cu en eco ded on epola iza ion o -80 mV a e a pulse o 40 mV las ing 6 ms. An exponen ial is i ed o he slow componen and ex apola ed back o he ins an o epola iza ion. Sub ac ion o he slow exponen- ial om he o al cu en yields a as componen on isola ion which could be i ed by ano he exponen ial wi h a as e ime cons an . Thus, each exponen ial unc ion ep esen s he closing ime cou se o slow (SD) and as (FD) deac i a ing Ca 2÷ on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Feb ua y 1, 1991 318 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 - 1991 ansien ac i a ion o SD channels and hei low ac i a ion ol age make some sep al neu ons capable o i ing ei he onically o in a bu s mode, depending on he memb ane po en ial o he cells (Al a ez de Toledo and L6pez-Ba neo, 1988). I is wo h men ioning ha ou p e ious da a al eady sugges ed ha in some sep al neu ons a signi ican p opo ion o FD channels migh be loca ed nea he soma. The cha ac e is ic slow, high h eshold Ca 2+ spikes o hese cells appea ed du ing small cu en injec ions, which is no a common inding in o he CNS neu ons, and i was ela i ely usual o ob ain eco dings whe e an ini ial Na + spike o small ampli ude was ollowed by la ge and b oade Ca ~+ ac ion po en ials. 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