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
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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.
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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
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[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 .
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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
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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.
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