The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons

The Rela ionship be ween Pulsa ile Sec e ion and Calcium Dynamics in Single, Li ing Gonado opin- Releasing Ho mone Neu ons* LUCI ´ANU ´N ˜EZ, CARLOS VILLALOBOS, F. R. BOOCKFOR, AND L. STEPHEN FRAWLEY Labo a o y o Molecula Dynamics, Depa men o Cell Biology and Ana omy, Medical Uni e si y o Sou h Ca olina, Cha les on, Sou h Ca olina 29425 ABSTRACT I is well es ablished ha pulsa ile elease o GnRH egula es he ep oduc i e axis, bu li le is known abou he mechanisms unde - lying his pulsa ili y. Recen indings ha GT1 cells, a line de i ed om he mouse emb yonic hypo halamus, elease GnRH in a pulsa- ile manne indica es ha his hy hmic ac i i y is an in insic p op- e y o GnRH neu ons. In se e al a emp s o unco e he in acel- lula basis o his pulsa ile phenomenon, i was e ealed ha in acellula calcium concen a ions change in a hy hmic ashion in GnRH neu ons and ha cellula depola iza ion, which igge s a sec e o y e en , is associa ed wi h p o ound calcium changes in he cells. These indings aised he in iguing possibili y ha pe iodic al e a ions in in acellula calcium concen a ions may unde lie he phenomenon o pulsa ile sec e ion in GnRH neu ons. To add ess his, we i s adap ed he use o FM1–43 luo escence o moni o changes o sec e ion in indi idual GT1–7 cells and hen combined his ap- p oach wi h simul aneous measu emen o in acellula ee calcium ([Ca 2⫹ ] i , u a 2 me hod). In ini ial alida ion expe imen s, we ound ha s imula ion o exocy osis wi h K ⫹ (75 mM)o N-me hyl-D-aspa - a e (NMDA, 100 ␮ M) p edic ably e oked dynamic inc eases o bo h FM1–43 and u a 2 luo escence. La e measu emen o calcium dy- namics and exocy o ic ac i i y in uns imula ed cells e ealed ha [Ca 2⫹ ] i unde wen ansi ions om quiescence o high oscilla o y beha io , and ha hese shi s we e equen ly associa ed wi h exo- cy o ic e en s. Mo eo e , hese calcium oscilla o y ansi ions and associa ed changes in sec e o y ac i i y occu ed synch onously among mos adjacen cells and a a equency simila o ha epo ed o pulsa ile elease o GnRH by en i e cul u es o GnRH neu ons. Taken oge he , hese esul s indica e ha he in insic sec e o y pulsa ili y o GnRH neu ons appea s o be a consequence o coo di- na ed, pe iodic changes in he pa e n o calcium oscilla ions wi hin indi idual cells. (Endoc inology 141: 2012–2017, 2000) THE PULSATILE sec e ion o GnRH se es as he d i ing o ce o he ep oduc i e axis by go e ning he pul- sa ile elease o gonado opic ho mones om he an e io pi ui a y in o he ci cula ion. The sou ce o hese GnRH pulses consis s o a ew housand neu ons (1) sca e ed in he p eop ic and mediobasal hypo halamic a eas (2). E en hough hese neu ons a e di use and do no appea o be highly o ganized mo phologically, i is qui e clea ha hey a e unc ionally in e connec ed as e idenced by he collec- i e pulsa ile elease o GnRH om he hypo halamus (3–7). Recen de elopmen by Mellon and co-wo ke s (8) o a con- inuous line o GnRH neu ons (GT1 cells) has p o ided a powe ul ool o aid in he elucida ion o he mechanism(s) unde lying pulse gene a ion. The GT1 cell line, de i ed om he mouse emb yonic hypo halamus, sha es mos o he unc ional cha ac e is ics o no mal GnRH neu ons. These ans o med cells elease GnRH in a pulsa ile ashion (9–12). Mo eo e , he equency a which GnRH pulses a e elabo- a ed by GT1 cells is qui e simila o ha epo ed in i o (5–7) and o p ima y cul u es o monkey emb yonic GnRH neu- ons (13). In ac , he pulsa ile elease o GnRH om cul u es o GT1 cells, which a e de oid o connec ion o any o he cell ypes, sugges s ha sec e o y pulsa ili y is an in insic p op- e y o he GnRH neu on (12, 14). Despi e he impo ance o his pulsa ili y in ep oduc ion, li le in o ma ion is a ailable conce ning he manne in which hese pulses a e gene a ed. Recen e idence sugges s ha calcium is a c i ical com- ponen in he ini ia ion and p opaga ion o hese pulses. Fi s , i was epo ed ha single GT1 cells exhibi spon aneous i ing o ac ion po en ials ha a e associa ed wi h oscilla ions o in acellula ee calcium ([Ca 2⫹ ] i ) (15). In e es ingly, he blockade o calcium channels by pha macological means o by emo al o ex acellula calcium, a maneu e ha inhib- i ed spon aneous elec ical ac i i y, abolished GnRH elease in GT1 cells (10, 15, 16). Simila ly, manipula ion o calcium le els in p ima y GnRH neu ons de i ed om emb yonic hesus monkeys also ma kedly dampened GnRH elease (13). These obse a ions indica e ha calcium is necessa y o GnRH sec e ion. A second line o e idence e eals a shi ing pa e n o in acellula ee calcium ha would be consis en wi h pulsa ile ac i i y in a cell popula ion. Cha les and Hale (17) ound ha , in some cases, GT1–7 cells exhibi spon a- neous oscilla ions o in acellula calcium ha appea ed o be synch onized among cells, sugges ing ha GnRH sec e o y pulsa ili y may ollow synch oniza ion o calcium oscilla- ions. Te asawa and co-wo ke s (18) also demons a ed a synch oniza ion o calcium oscilla ions in cul u ed GnRH Recei ed No embe 24, 1999. Add ess all co espondence and eques s o ep in s o: D . L. S e- phen F awley, Labo a o y o Molecula Dynamics, Depa men o Cell Biology and Ana omy, Medical Uni e si y o Sou h Ca olina, Cha les- on, Sou h Ca olina 29425. E-mail: [email p o ec ed]. * This wo k was suppo ed by NIH G an s DK-38215 and HD-37657 ( o L.S.F.) and a pos doc o al ellowship om Minis e io de Educacio´n y Cul u a o Spain ( o L.N.). These esul s we e p esen ed in pa a he 81 s Annual Mee ing o The Endoc ine Socie y, June 1999, San Diego, Cali o nia (Abs ac P1–503). 0013-7227/00/$03.00/0 Vol. 141, No. 6 Endoc inology P in ed in U.S.A. Copy igh © 2000 by The Endoc ine Socie y 2012 Downloaded om h ps://academic.oup.com/endo/a icle/141/6/2012/2988424 by Biblio eca Uni e si a ia.Facul ad de Medicina use on 19 Janua y 2021 neu ons om emb yonic hesus monkeys. Thus, in addi ion o he ole ha calcium may play in he expo o GnRH, i appea s ha he oscilla o y pa e n o his componen is qui e consis en wi h he unique hy hmic ac i i y ha is he hall- ma k o GnRH neu onal unc ion. A emp s o u he elu- cida e a ela ionship be ween pulses o GnRH elease and in acellula calcium dynamics has been hinde ed by he lack o echnology enabling he measu emen o bo h calcium and sec e ion in he same, li ing neu ons. In he p esen s udy, we acqui ed his echnology by combining digi al-imaging luo escence mic oscopy o u a-2 ( o moni o calcium changes) wi h con inuous measu emen o luo escence emi ed a e exci a ion o FM1–43 ([N-(3- ie hylammonio- p opyl)-4-(p-dibu ylaminos y yl) pi idinium dib omide]), a memb ane- u no e p obe. FM1–43 is ela i ely non luo es- cen in aqueous solu ion, bu becomes highly luo escen in he lipid en i onmen o cell memb anes ( he quan um yield o FM1–43 inc eases up o 350- old; Re . 19). Endocy ocis, which in a iably ollows exocy o ic elease om cells, esul s in he in e naliza ion o he p obe and he inc ease o luo- escence emission a e exci a ion (20). These combined ap- p oaches we e used in he ollowing expe imen s o de e - mine he ela ionship be ween calcium changes and he pulsa ile elease o GnRH in GT1–7 cells. Ma e ials and Me hods Cell cul u e GT1–7 cells (kindly p o ided by Richa d I. Weine , Uni e si y o Cali o nia a San F ancisco) we e g own in DMEM (Li e Technologies, Inc., Gai he sbu g, MD) wi h 10% FBS, 100 IU/ml penicillin, 100 ␮ g/ml s ep omycin, and 0.25 ␮ g/ml ungizone. The cul u es we e main ained in a wa e -sa u a ed a mosphe e o 95% ai and 5% C0 2 . Fo expe imen s, cells we e pla ed (a 10 5 cells/ml) on o poly-l-lysine (0.01 mg/ml) coa ed glass co e slips and cul u ed o 4 o 6 days be o e luo escence measu emen s. Fluo escence mic oscopy o FM1–43 FM1–43 up ake was measu ed essen ially as epo ed p e iously by Sho e e al. (21). B ie ly, GT1–7 cells we e washed wi h imaging medium (22) composed o a mix u e o Medium 199 and Nu ien mix u e F-12 [1:1] (Li e Technologies, Inc.) in which sodium bica bona e had been eplaced by HEPES bu e . The co e slips we e moun ed in a Sykes- Moo e chambe (23) and placed unde he mic oscope (Ca l Zeiss Ax- io e 135 TV, Jena, Ge many) in a empe a u e-con olled chambe (37 C). Then, cells we e exposed o 10 min o he memb ane- u no e p obe FM1–43 (2 ␮ m, Molecula P obes, Inc., Eugene, OR). Finally, cells we e epi-illumina ed wi h 490 nm ligh (100 msec e e y 20 sec) h ough a Ca l Zeiss Fluo 40⫻1.3 n.a. objec i e using a ibe op ic ligh sc amble (Technical Video, Woods Hole, MA) and a 500 nm dich oic mi o . The ligh emi ed was passed h ough a ba ie il e (515 nm long pass, Ch oma Technologies, B a lebo o, VT) and was collec ed con inuously a he co esponding imes by a cooled CCD came a (Hamama su Pho- onics, Hamama su, Japan). The luo escen signal o FM1-43 loaded cells was e y in ense, and he exci a ion ligh was educed by use o neu al densi y il e s (Ch oma Technologies). Fluo escen images we e s o ed and analyzed using he Me a luo so wa e om Uni e sal Im- aging (Wes Ches e , PA). Combina ion o FM1–43 luo escence and [Ca 2⫹ ] i measu emen s Fo combina ion o FM1–43 luo escence measu emen s and digi al imaging luo escence mic oscopy o u a-2, we used he p o ocol de- eloped by Sho e e al. (21) wi h mino modi ica ions. In b ie , GT1–7 FIG. 1. Digi al imaging luo escence mic oscopy o FM1–43 in indi- idual GT1–7 cells. GT1–7 cells we e exposed con inuously o he memb ane- u no e p obe FM1–43 (2 ␮ M) and 10 min la e subjec ed o ligh exci a ion. Ligh emi ed abo e 515 nm was eco ded be o e (A) and 10 min a e (B) addi ion o he glu ama e ecep o agonis NMDA (100 ␮ M), a well-known s imula o o GnRH elease. No e he apid inc ease in luo escence in ensi y (indica i e o sec e ion) a e ea men . FIG. 2. Moni o ing o s imulus-induced memb ane e ie al wi h FM1–43. Cells p ocessed as in Fig. 1 we e s imula ed o sec e e wi h high-K ⫹ medium o NMDA. Rep esen a i e examples o esponses o indi idual cells (#1–4) eco ded in he same mic oscopic ields a e shown. Also shown a e he a e aged aces (A g; mean ⫾SEM) o all cells p esen in each o he mic oscopic ields. Da a a e ep esen a i e o 66 (K ⫹ ) and 50 cells (NMDA) s udied in 3 independen expe imen s. These esul s demons a e ha s imula ion o sec e ion in GT1–7 cells e okes he up ake o he memb ane- u no e p obe FM1–43, and ha his p ocess can be moni o ed con inuously. CALCIUM OSCILLATIONS AND SECRETION 2013 Downloaded om h ps://academic.oup.com/endo/a icle/141/6/2012/2988424 by Biblio eca Uni e si a ia.Facul ad de Medicina use on 19 Janua y 2021 cells we e washed wi h imaging medium and loaded wi h u a-2 by incuba ing hem o abou 60 min a oom empe a u e wi h u a-2/AM (5 ␮ m, Molecula P obes, Inc., Eugene, OR). Cells we e hen moun ed in a chambe as desc ibed abo e and incuba ed unde he mic oscope o 10 min wi h FM1–43 (2 ␮ m). Cells kep in FM1–43 con aining medium we e hen epi-illumina ed al e na ely wi h exci a ion ligh a 340 nm and 380 nm o 100 msec e e y 5 sec and a 490 nm o 300 ms e e y 50 sec. This was achie ed by passing he exci a ion ligh h ough an al e na ing wheel con aining 10 nm (340, 380 o u a-2) o 5 nm (490 o FM1–43) band-pass in e e ence il e s. In his p o ocol, we used a 400 nm dich oic mi o . Emi ed ligh was passed h ough a ba ie il e (515 nm long pass) and collec ed con inuously a he co esponding imes as desc ibed abo e. The images we e s o ed and analyzed using Me a luo so wa e. Pixel by pixel a ios o consecu i e ames ob ained a 340 and 380 nm exci a ion we e p oduced and [Ca 2⫹ ] i alues we e es ima ed by com- pa ison wi h u a-2 s anda ds (24). In expe imen s using FM1–43 alone o in combina ion wi h u a 2, we did no obse e any dele e ious e ec du ing p olonged exposu e o loaded cells o exci a ion ligh . Fo long- e m expe imen s, he chambe was closed o keep condi ions s able and a oid changes in medium concen a ion due o e apo a ion. In con as , he chambe was opened o sho - e m expe imen s o acili a e addi- ion o ea men s. K ⫹ , NMDA and nimodipine we e added a inal concen a ions o 75 mm, 100 ␮ m, and 100 mm, espec i ely. Each agen was es ed by addi ion o 40 ␮ l o a mo e concen a ed solu ion (also con aining 2 ␮ mFM1–43) in o he open chambe con aining 800 ␮ lo imaging medium. The chambe s ha included s imula ing agen s we e imaged immedia ely. Those con aining nimodipine we e incuba ed o 10 min be o e imaging. Each agen emained in con ac wi h he cells o he en i e cou se o he expe imen . Resul s and Discussion As a i s s ep, we had o de elop a me hod o con inu- ously moni o ing sec e o y ac i i y wi hin indi idual GnRH neu ons. In pu sui o his, we assessed whe he he mem- b ane- u no e p obe FM1–43 could be used o his pu - pose. This dye is non luo escen in aqueous solu ion, bu becomes luo escen upon inco po a ion in o he lipid en i- onmen o he plasma memb ane (19). Du ing a sec e o y e en , g anule esicles use wi h he plasma memb ane ex- posing mo e memb ane su ace a ea o he dye. Molecules o FM1–43 ha ha e inse ed be ween phospholipids in he ou e lea le o he plasma memb ane hen en e he cell h ough endocy osis as he esicle memb anes a e ecycled. Collec i ely, he ex ensi e memb ane ecycling ha occu s ollowing a sec e o y e en inc eases he le el o luo escence in he cell. Figu e 1 p o ides a pho omic og aph o GT1–7 cells imaged in he p esence o FM1–43 (2 ␮ m) and subse- quen ly exposed o exci a ion ligh . Fluo escence images o he cells we e eco ded be o e (Fig. 1A) and a e (Fig. 1B) exposu e o he glu ama e ecep o agonis N-Me hyl d- aspa a e (NMDA), a well-es ablished s imula o o GnRH sec e ion. As shown, he dye appea ed o be dis ibu ed o e bo h he soma and neu i es o he GT1–7 cells unde basal condi ions. In mos o he cells, an inc ease in memb ane inco po a ion o he p obe ollowed s imula ion, as e i- denced by he local inc ease o luo escence in ensi y a e ea men . The esponsi eness o luo escen signal o s im- ula ion is illus a ed mo e quan i a i ely in Fig. 2 o NMDA (100 ␮ m, panel A) as well as o high K ⫹ (75 mm, panel B) medium. The la e ea men is used commonly o depo- la ize cells and he eby causes he exocy osis o s o ed se- c e o y p oduc . Examples o esponses om indi idual cells (#1–4) o he a e aged aces (A g.; mean ⫾sem) a e shown. Clea ly, each o hese ea men s ini ia ed a ise in luo es- cence wi hin minu es o adminis a ion, consis en wi h he ime- ame epo ed o he endocy osis ha occu s subse- quen o exocy osis in neu ons (19). Addi ion o NMDA s imula ed memb ane e ie al in 92% o he cells (n ⫽50, 3 independen expe imen s). T ea men wi h high-K ⫹ me- dium esul ed in an inc ease o FM1–43 luo escence in 92% o he cells s udied (n ⫽66 cells, 3 independen expe imen s). Ou demons a ion ha unequi ocal inc eases o FM1-43 luo escence occu ed subsequen o s imula ions wi h agen s known o elici s ong sec e o y esponses se es o con i m he alidi y o his p obe o making dynamic mea- su emen s o sec e ion in GT1–7 cells. To explo e he ela ionship be ween in acellula calcium dynamics and sec e o y ac i i y in he same GT1–7 cells, we adop ed a no el s a egy de eloped by Sho e e al. (21) o use on an e io pi ui a y cells. I consis s o loading cells wi h bo h he calcium sensi i e p obe u a-2/AM and he mem- b ane e ie al p obe FM1–43. The cells a e hen subjec ed o a iome ic de e mina ion o [Ca 2⫹ ]i (a e dual exci a ion a 340 and 380 nm) ollowed by moni o ing o FM1-43 inco - po a ion a e exci a ion a 490 nm. Figu e 3 shows an ex- ample o he images o FM1–43 luo escence (panel A) and in acellula calcium (340/380 a ios coded in pseudocolo , panel B) eco ded almos simul aneously. As illus a ed, his app oach allows analysis o bo h sec e o y ac i i y and FIG. 3. Combina ion o FM1–43 and Fu a-2 luo escence measu emen s. GT1–7 cells we e loaded wi h u a-2/AM o 1 h and hen exposed con inuously o FM1–43. Ten minu es la e , cells we e sequen ially epi-illumina ed wi h 340, 380, and 490 nm exci a ion ligh and luo escence emissions (abo e 515 nm) esul ing om each exci a ion we e eco ded o e ime. Ligh emi ed a e exci a ion a 490 nm e lec s FM1–43 up ake (A). Ra ios o emissions esul ing om exci a ions a 340 and 380 nm we e used o es ima e [Ca 2⫹ ] i . He e (B) hey a e ep esen ed by pseudocolo images. Rela i e [Ca 2⫹ ]i is indica ed on he ascending pseudocolo scale o he igh in which wa me colo s e lec highe a ios. 2014 CALCIUM OSCILLATIONS AND SECRETION Endo •2000 Vol 141 •No 6 Downloaded om h ps://academic.oup.com/endo/a icle/141/6/2012/2988424 by Biblio eca Uni e si a ia.Facul ad de Medicina use on 19 Janua y 2021 [Ca 2⫹ ] i in he same neu ons. Wi h he goal o alida ing his combina o ial app oach, we s imula ed GT1–7 cells wi h ei- he high-K ⫹ medium o NMDA as be o e and eco ded bo h o he abo e men ioned a iables. As illus a ed in Fig. 4, A–C, s imula ion wi h high K ⫹ e oked an inc ease o [Ca 2⫹ ] i (ligh e lines), which was ollowed sho ly he ea e by exo- cy osis, as e ealed by he inc ease o FM1–43 luo escence (bold lines). We ound ha 89% o cells s imula ed wi h his high-K ⫹ medium (n ⫽62, 3 expe imen s) esponded wi h a [Ca 2⫹ ] i inc ease and 85% o hese esponde s exhibi ed a ise in FM1–43 luo escence. As u he illus a ed in Fig. 4, D–F, s imula ion wi h NMDA also inc eased [Ca 2⫹ ] i and induced memb ane e ie al in he cells imaged. This occu ed in 93% o he cells s udied (n ⫽60, 3 expe imen s). In hose ew ins ances in which he e we e no [Ca 2⫹ ]i esponses ollowing s imula ion, he e was also a lack o inc ease in FM1–43 luo escence (da a no shown). Taken oge he , hese da a demons a e he easibili y o making essen ially simul a- neous measu emen s o calcium dynamics and sec e o y e en s in he same li ing GT1–7 neu on. Ha ing es ablished he alidi y and u ili y o using FM1–43 o measu e sec e ion alone o in combina ion wi h u a-2 imaging o calcium, we hen se ou o es ablish he ela ionship be ween hese a iables unde basal condi ions. In p elimina y expe imen s, we measu ed FM1–43 luo es- cence alone and ound ha oughly hal o he cells (52%, n ⫽ 56 cells) exhibi ed spon aneous changes in sec e ion o e ime and ha hese had an a e age equency o 1.1 ⫾0.1 pulses/h consis en wi h ha epo ed o GnRH elease om pe used GT1 cul u es (10, 11). This beha io o FM1–43 FIG. 4. Simul aneous measu emen s o sec e agogue-induced changes o in acellula calcium and memb ane e ie al in indi idual GT1–7 cells. Cells p ocessed as in Fig. 4 we e s imula ed wi h high-K ⫹ medium (le panel) o he glu ama e ecep o agonis NMDA ( igh panels). S imula ion induced an inc ease o [Ca 2⫹ ] i (ligh e lines), which was ollowed sho ly he ea e by exocy osis, as e ealed by he inc ease in FM1–43 luo escence (bold lines). Rep esen a i e examples o esponses ob ained om indi idual cells (A, B, D, E) eco ded in he same mic oscopic ields a e shown. Da a a e ep esen a i e o 62 and 60 cells, espec i ely, s udied in h ee independen expe imen s o each agonis . CALCIUM OSCILLATIONS AND SECRETION 2015 Downloaded om h ps://academic.oup.com/endo/a icle/141/6/2012/2988424 by Biblio eca Uni e si a ia.Facul ad de Medicina use on 19 Janua y 2021 luo escence is illus a ed in Fig. 5 by ep esen a i e example in adjacen cells (panels A and B) and he a e aged aces o all cells in he same mic oscopic ield (panel C). When cal- cium imaging was combined wi h analysis o sec e ion, we ound ha a change in FM1–43 luo escence (indica ing a sec e o y e en ) was always accompanied by he esump ion o high le el calcium oscilla ions a e a pe iod o ela i e quiescence. In hese expe imen s, changes o bo h sec e o y ac i i y and mode o [Ca 2⫹ ] i oscilla ions occu ed a an a - e age a e o 0.96 ⫾0.15 (mean ⫾sem,n⫽59) pulses pe hou , simila o ha ound in ou p elimina y expe imen s on sec e ion alone. O he in es iga o s ha e epo ed ha GT1 cells o cells in p ima y hypo halamic cul u es exhibi spon- aneous changes o calcium oscilla o y p o iles. Bo h Cha les and Hales (17) and Spe gel e al. (15) demons a ed he p es- ence o spon aneous calcium oscilla ions in GT1–7 cells, which we e associa ed wi h i ing o spon aneous ac ion po en ials. Recen ly, he p esence o pe iodic calcium oscil- la ions was also demons a ed in indi idual p ima y neu- ons om monkeys (18). In each o hese s udies, i was p oposed ha changes in calcium oscilla o y ac i i y we e associa ed wi h he hy hmic elease o GnRH, bu a di ec demons a ion o he ela ionship be ween sec e ion and cal- cium oscilla ions was no a emp ed. Ou indings demon- s a e ha shi s occu in he o e all oscilla o y s a es o calcium and ha hese shi s a e indeed linked o sec e o y e en s a a equency consis en wi h ha o GnRH pulsa- ili y. Al hough all sec e o y e en s in ou s udies we e ac- companied by calcium shi s o a highe oscilla o y s a e, we ound ha no all shi s in he unc ional s a e o calcium we e linked o sec e o y episodes (62%, n ⫽69). Thus, calcium oscilla o y ansi ions appea o be necessa y, bu no su i- cien , o induc ion o GnRH elease. The e a e se e al pos- sible explana ions o his. One possibili y is ha a sec e o y e en does occu in conce wi h e e y shi o a highe oscilla o y s a e, bu he sec e o y bu s s a e no o su icien magni ude o be de ec ed wi h ou sys em. Ano he expla- na ion may be ela ed o he di e ences in eadiness o sec e ion om cell o cell. Some cells may con ain abundan sec e o y g anules and a e able o espond o an app op ia e s imulus o sec e ion such as a calcium oscilla o y ansi ion whe eas o he s a e no , e en hough such in acellula cal- cium signaling occu s. Whe he o no hey sec e e, mos o hese cells do exhibi shi s in [Ca 2⫹ ] i oscilla o y beha io (76%, n ⫽91) e ealing an in e nal hy hm in he majo i y o cells in cul u e. Ou esul s ob ained om analysis o mul iple cells in he same mic oscopic ields u he indica e ha in e cellula as well as in acellula signaling occu s wi h espec o calcium oscilla o y and GnRH sec e o y ac i i ies. We ound ha adjacen cells usually (76%, n ⫽69) exhibi ed coo dina ed shi s in calcium oscilla o y beha io . Mo eo e , he majo i y o sec e o y e en s (80%, n ⫽51) occu ed in a synch onized manne among adjacen cells. In ac , we obse ed ha 100% o he synch onic ac i i y in sec e ion was associa ed wi h synch onic shi s in calcium oscilla o y s a es, sugges ing a causal ela ionship be ween hese pa ame e s. This possibil- i y was con i med when ea men wi h nimodipine (100 nm), a calcium channel blocke , abolished bo h pe iodic in- c eases in FM1–43 luo escence and spon aneous [Ca 2⫹ ] i os- FIG. 5. Rela ionship be ween calcium and sec e o y dynamics in GT1–7 cells. Cells we e subjec ed o simul aneous moni o ing o bo h [Ca 2⫹ ] i (lowe lines) and sec e o y ac i i y (FM1–43 luo escence, uppe lines) o 2 h. The beha io o ep esen a i e adjacen cells (A and B) and he a e age esponses o all cells in he same mic oscopic ield (C) a e shown he e. Ve ical lines ep esen synchonic sec e o y episodes. This expe - imen is ep esen a i e o h ee ou o i e in which we ound synch onic sec e o y ac i i y (n ⫽59 cells, h ee expe imen s). Shown in Panel D is he e ec o nimodipine ea men on calcium oscilla ions and sec e o y ac i i y (n ⫽43 cells, h ee expe imen s). 2016 CALCIUM OSCILLATIONS AND SECRETION Endo •2000 Vol 141 •No 6 Downloaded om h ps://academic.oup.com/endo/a icle/141/6/2012/2988424 by Biblio eca Uni e si a ia.Facul ad de Medicina use on 19 Janua y 2021 cilla ions (Fig. 5D). The mechanism by which his coo dina- ion is e ec ed emains o be es ablished. The possibili y ha GnRH neu ons may communica e h ough synap ic ans- mission (25), gap junc ions (26), o by elease o di usable subs ances (27, 28) has been unde ex ensi e in es iga ion. When aken oge he , ou esul s in i e he de elopmen o a wo king hypo hesis o explain he gene a ion o in insic sec e o y pulses in GT1–7 cells. I is clea om ou indings ha changes in oscilla o y pa e ns and exocy o ic e en s a e closely linked in a empo al ashion. Fo he elabo a ion o a sec e o y pulse, i appea s ha ela i ely quiescen cells be- gin o display a cha ac e is ic pa e n o high- equency, high ampli ude calcium oscilla ions. These oscilla ions a e com- munica ed and synch onized among adjacen cells and esul in synch onized exocy o ic e en s. The e o e, i is emp ing o specula e ha hese igh ly coupled e en s la gely com- p ise he basis o he elusi e “GnRH pulse gene a o .” Acknowledgmen s We hank William J. Faugh and Da id C. Leaumon o expe echnical assis ance. Re e ences 1. 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