Blockade o Neu onal Facili a o y
Nico inic Recep o s Con aining ␣3␤2
Subuni s Con ibu e o Te anic Fade
in he Ra Isola ed Diaph agm
MIGUEL FARIA, LAURA OLIVEIRA, M. ALEXANDRINA TIMO
´TEO,
M. GRAC¸ A LOBO, AND PAULO CORREIA-DE-SA
´
Labo a o´ io de Fa macologia, Unidade Mul idisciplina de In es igac¸a˜o Biome´dica (UMIB), Ins i u o de Cieˆncias
Biome´dicas de Abel Salaza (ICBAS), Uni e sidade do Po o, 4099-003 Po o, Po ugal
KEY WORDS neu omuscula junc ion; ace ylcholine elease; sa e y ac o ; muscle
elaxan s
ABSTRACT Nico inic ecep o (nAChR) sub ypes in ol ed in p e- and pos junc ional
ac ions unde lying e anic ade we e s udied in a ph enic-ne e hemidiaph agms. We
in es iga ed he abili y o sub ype-specific nAChR an agonis s o dep ess ne e-e oked
con ac ions and [
3
H]-ace ylcholine ([
3
H]-ACh) elease. Muscle ension was ansien ly
inc eased du ing b ie high equency ains (50 Hz o 5 sec). The ank po ency o de o
nAChR an agonis s o educe e anic peak ension was ␣-bunga o oxin ⬎d- ubocu a ine
Ⰷmecamylamine ⬎hexame honium. Reduc ion o maximal e anic ension p oduced by
dihyd o-␤-e y h oidine (0.03–10 ␮M), me hyllycaconi ine (0.003–3 ␮M), and ␣-cono oxin
MII (0.001–0.3 ␮M) did no exceed 30%. Besides educ ion o peak ension d- ubocu a-
ine (0.1–0.7 ␮M), mecamylamine (0.1–300 ␮M), and hexame honium (30–3,000 ␮M)
also caused e anic ading. Wi h ␣-cono oxin MII (0.001–0.3 ␮M) and dihyd o-␤-e y h-
oidine (0.03–10 ␮M), e anic ade was e iden only a e dec easing he sa e y ac o o
neu omuscula ansmission (wi h high magnesium ions, 6–7 mM). The an agonis ank
po ency o de o educe e oked (50 Hz o 5 sec) [
3
H]-ACh elease om mo o ne e
e minals was ␣-cono oxin MII (0.1 ␮M) ⬎dihyd o-␤-e y h oidine (1 ␮M) ⬃d- ubocu-
a ine (1 ␮M) ⬎mecamylamine (100 ␮M) ⬎hexame honium (1,000 ␮M). When applied
in a concen a ion (0.3 ␮M) abo e ha p oducing e anic pa alysis, ␣-bunga o oxin
ailed o a ec [
3
H]-ACh elease. Da a ob ained sugges ha pos junc ional neu omus-
cula elaxan s in e ac wi h ␣-bunga o oxin-sensi i e nico inic ecep o s con aining
␣1-subuni s, whe eas blockade o neu onal ␣3␤2-con aining ecep o s p oduce e anic
ade by b eaking nico inic au o acili a ion o ace ylcholine elease. Synapse 49:77–88,
2003. ©2003 Wiley-Liss, Inc.
INTRODUCTION
In myog aphic eco ds, neu omuscula ade, e anic
ade, o Wedensky inhibi ion is he inabili y o a muscle
o sus ain ension du ing high equency (30–80 Hz)
mo o ne e s imula ion in he p esence o muscle e-
laxan s such as ubocu a ine (d-TC) (Bowman, 1980;
an de Kloo and Molgo´, 1994). In elec ophysiological
expe imen s, undown o endpla e po en ials is also
obse ed du ing high- equency s imula ion ains, an
e ec esul ing om dec eases in he quan al ou pu
(Ma zne e al., 1988) wi hou significan changes in
endpla e es ing po en ials (Magleby e al., 1981). Ion-
opho e ic pulses o ace ylcholine (ACh) deli e ed a a
equency o 50 Hz did no p oduce e anic ade ei he
in he absence o in he p esence o d-TC (Gibb and
Ma shall, 1986). Mo eo e , hese au ho s demon-
s a ed ha depola iza ion o muscle fibe s by ion o-
pho e ically applied ACh could s ill be obse ed ollow-
ing ading o ne e-e oked endpla e po en ials (Gibb
and Ma shall, 1984). Thus, i was sugges ed ha
wi ch blockade and e anic ade a e sepa a e and in-
Con ac g an sponso : FCT; Con ac g an numbe s: POCTI/36545/FCB/
2000 and UMIB-215/94.
*Co espondence o: P. Co eia-de-Sa´, MD, PhD, Labo a o´ io de Fa macologia,
Ins i u o de Cieˆncias Biome´dicas de Abel Salaza (ICBAS)—Uni e sidade do
Po o, L. P o . Abel Salaza , 2, 4099-003 Po o, Po ugal.
E-mail: [email p o ec ed].
Recei ed 8 Oc obe 2002; Accep ed 4 Ma ch 2003
DOI 10.1002/syn.10211
SYNAPSE 49:77–88 (2003)
©2003 WILEY-LISS, INC.
dependen ac ions; wi ch ension dep ession is linked
o he compe i i e block o pos synap ic nico inic ecep-
o s (nAChRs), whe eas endpla e undown o e anic
ade esul p edominan ly om a p ejunc ional ac ion
o nAChRs an agonis s (Gibb and Ma shall, 1986).
ACh may enhance i s own elease du ing a pe iod o
epe i i e mo o ne e ac i i y by ac i a ing nico inic
au o ecep o s. The posi i e nico inic eedback mecha-
nism was sugges ed om s udies measu ing mechani-
cal ension and elec ophysiological signals in he p es-
ence o nico inic an agonis s (see o a e iew, Bowman
e al., 1988). In addi ion, da a om adiolabeled expe -
imen s showed ha he nico inic agonis 1,1-dime hyl-
4-phenylpipe azinium ma kedly inc eased, while nico-
inic an agonis s dec eased, he elease o [
3
H]-ACh
elease in esponse o ne e s imula ion (Vizi and So-
mogyi, 1989; Wessle , 1989; Co eia-de-Sa´ and Ribei o,
1994). Nico inic au o acili a ion is clea ly equency-
dependen and may ope a e as a empo a y p esynap-
ic amplifie o inc ease he sa e y ac o o ansmis-
sion, pa icula ly when inc eased muscula s eng h is
needed (Waud and Waud, 1971; Singh and P io , 1998;
Wood and Sla e , 2001).
Nico inic ecep o s a e a he e ogeneous amily. Di-
e si y in nAChR ypes is de i ed om he a iabili y
o genes encoding o ecep o subuni s and hei pen-
ame ic subuni combina ions (namely, associa ions
be ween ␣1–␣9 and ␤1–␤4 subuni s) (Lukas e al.,
1999). Subuni s con e dis inc s uc u al and unc-
ional p ope ies (e.g., calcium pe meabili y, desensi i-
za ion a e, phospho yla ion sensi i i y) o he nAChR
ypes ha hey o m. Se e al lines o e idence indica e
ha neu onal and muscula nico inic ecep o s p esen
a he neu omuscula junc ion di e in hei pha ma-
cological p ofiles (e.g., Gibb and Ma shall, 1984; 1986;
Vizi e al., 1987). Howe e , hese findings a e no ye
clea ly in eg a ed wi h molecula defini ions o he e-
cep o sub ypes ha pa icipa e in p e- and pos junc-
ional ac ions unde lying changes o neu omuscula
ansmission. The muscle endpla e nAChR is bes
cha ac e ized and has an (␣1)
2
(␤1)ε␦subuni configu-
a ion (Schue ze and Role, 1987; Salpe e e al., 1988).
In con as , he cu en ally o neu onal nAChR sub-
uni s in mammals is ele en (␣2–␣7, ␣9, ␣10, ␤2–␤4)
wi h an addi ional subuni , ␣8, iden ified in a ian spe-
cies.
The p esen wo k was designed o s udy he ole o
acili a o y nico inic au o ecep o s block on e anic
ade and o in es iga e he ype o nAChR ha migh
be in ol ed in i s ope a ion. Fo his pu pose we es ed
he e ec s o se e al nico inic an agonis s, d- ubocu a-
ine (d-TC), hexame honium (HEX), mecamylamine
(Meca), dihyd o-␤-e y h oidine (DH-␤-E), me hyllyca-
coni ine (MLA), ␣-bunga o oxin (BTX), and ␣-cono-
oxin MII (CTX MII) on ension esponses and [
3
H]-
ACh elease igge ed by b ie high- equency ains
(50 Hz o 5 sec) deli e ed o he a ph enic ne e-
hemidiaph agm p epa a ions. Like Meca, HEX was
also fi s ecognized as a ganglionic nAChR-blocking
agen ; hese agen s exe hei e ec s ac ing as nico-
inic channel blocke s and a e conside ed noncompe i-
i e an agonis s. DH-␤-E is a compe i i e neu onal nic-
o inic an agonis wi h a deg ee o selec i i y o
ecep o s con aining ␣4␤2 and ␣3␤2 subuni s (Cha ez-
No iega e al., 1997). The Delphinium alkaloid, MLA,
compe i i ely an agonizes ␣7 nAChRs (K
i
⬃1 nM) and,
unlike BTX, disc imina es be ween neu onal ␣7 and
muscle- ype ␣1 ecep o s. BTX was ins umen al in he
isola ion and pu ifica ion o muscula ␣1-con aining
nAChRs, bu i is also a highly po en and selec i e
an agonis a ␣7 nAChRs (K
1
⬃1 nM) wi hou in e -
ac ing wi h ␣/␤he e ome s ( o a e iew, see Dwoskin
and C ooks, 2001). CTX MII, a 16- esidue polypep ide
om he enom o he pisci o ous cone snail Conus
magus, has a high deg ee o selec i i y o ␣3␤2-con-
aining neu onal nAChRs (Ca ie e al., 1996), al-
hough i migh also block ecep o s con aining ␣6 sub-
uni s wi h a high a fini y (e.g., Ku ya o e al., 2000).
MATERIALS AND METHODS
Ra s (Wis a , 150–200 g) o ei he sex (Cha les
Ri e , Ba celona, Spain) we e kep a a cons an em-
pe a u e (21°C) and a egula ligh (06.30–19.30 h)
da k (19.30–06.30 h) cycle wi h ood and wa e ad
libi um. The animals we e killed by s unning ollowed
by exsanguina ion. Animal handling and expe imen s
ca ied ou a ICBAS ollowed he guidelines o he
In e na ional Council o Labo a o y Animal Science
(ICLAS). The expe imen s we e ca ied ou in i o on
le ph enic ne e-hemidiaph agm p epa a ions (4–6
mm wid h). Each muscle was supe used wi h Ty ode’s
solu ion (pH 7.4) wi h he ollowing composi ion (mM):
NaCl 137, KCl 2.7, CaCl
2
1.8, MgCl
2
1, NaH
2
PO
4
0.4,
NaHCO
3
11.9, glucose 11.2, and choline 0.001, a 37°C.
This solu ion was con inuously gassed wi h a mix u e
o O
2
(95%) and CO
2
(5%).
Ne e s imula ion condi ions
The le ph enic ne e was s imula ed wi h an ex a-
cellula glass-pla inum suc ion elec ode placed nea
i s fi s di ision b anch, o a oid di ec s imula ion o
muscle fibe s (indi ec s imula ion). To e alua e d ug
Abb e ia ions
ACh ace ylcholine
BTX ␣-bunga o oxin
CTX GIIIB ␮-cono oxin GIIIB
CTX MII ␣-cono oxin MII
DH-␤-E dihyd o-␤-e y h oidine
HEX hexame honium
Meca mecamylamine
MLA me hyllycaconi ine
MT-7 musca inic oxin 7
nAChRs nico inic ace ylcholine ecep o s
d-TC d- ubocu a ine
78 M. FARIA ET AL.
e ec s on muscle con ac ile p ope ies, di ec s imu-
la ion o muscle fibe s was deli e ed h ough a pai o
pla inum elec odes placed a each side o he dia-
ph agm nea i s cos al inse ion (field s imula ion).
Sup amaximal in ensi y (cu en s eng h o 8 mA),
ec angula pulses o 0.04 ms (indi ec s imula ion), o
1-ms (field s imula ion) du a ion we e used o achie e
fi ing synch oniza ion, hus educing he numbe o
silen uni s (mo oneu ons and/o muscle fibe s) ha
migh make in e p e a ion o da a di ficul . The pulses
we e deli e ed by a G ass S48 (Quincy, MA, USA)
s imula o coupled o a s imulus isola ion uni (G ass
SIU5) ope a ing in a cons an cu en mode. The s im-
ula ion pa ame e s we e con inuously moni o ed on an
oscilloscope (Megu o, MO-1251A, Japan) and we e
wi hin he same ange used in p e ious s udies wi h
his p epa a ion (e.g., Wessle and Kilbinge , 1986;
Co eia-de-Sa´ e al., 2000).
Muscle ension eco dings
When eco ding ension esponses, he inne a ed
diaph agm s ips we e moun ed e ically in a con en-
ional 10-ml capaci y isola ed o gan ba h chambe .
Te ani (5 sec long) deli e ed wi h a equency o 50 Hz
we e applied once e e y 15 min. Di ec - and ne e-
induced e anic esponses we e eco ded isome ically
a a es ing ension o 50 mN wi h a o ce ansduce
and displayed on a Hugo-Sachs (Ge many) eco de .
A e he ini ial s abiliza ion pe iod, hese expe imen-
al condi ions allowed a well-p ese ed e anic pa e n
o se e al hou s in he absence o es d ugs. Solu ions
we e changed ans e ing he inle ube o he pe i-
s al ic pump (Gilson, Minipuls3, F ance) om one flask
o ano he . The flow a e was 20 ml min
⫺1
du ing he
fi s min a e changing he solu ions and 5 ml min
⫺1
un il he nex changeo e o solu ions. Tes d ugs we e
allowed o con ac wi h he p epa a ions a leas 12
min be o e e anus; incuba ion ime wi h BTX was
p olonged o 45 min in some o he expe imen s. The
ension p oduced a he beginning o e anic s imula-
ion (a) was compa ed wi h ha ob ained a he end o
e anic s imula ion (b) (c . Sil a e al., 1999). The a io
R(R⫽b/a) ob ained a e d ug addi ion was aken as
a pe cen age o ha obse ed be o e any d ug admin-
is a ion. Ze o pe cen ep esen s equali y be ween a-
ios. Posi i e and nega i e alues ep esen inc emen
and ading o he e anic ension, espec i ely. In o de
o educe he sa e y ma gin o neu omuscula ans-
mission (Pa on and Waud, 1967; Wood and Sla e ,
2001), MgCl
2
(6–7 mM) was added o he ba h in some
o he expe imen s. Osmola i y was main ained by
equimola subs i u ion o NaCl. Ele a ion o magne-
sium ions o 6 and 7 mM dec eased he ampli ude o
ne e-e oked e anic esponses by 36 ⫾5% (n⫽5) and
74 ⫾6% (n⫽4), espec i ely.
Iso ope expe imen s
The p ocedu es used o labeling he p epa a ions
and measu ing e oked [
3
H]-ACh elease we e p e i-
ously desc ibed (Co eia-de-Sa´ e al., 1991) and used
wi h mino modifica ions. Expe imen s we e pe -
o med in he absence o cholines e ase inhibi o s o
p e en unphysiological ex acellula accumula ion o
ACh. Ph enic ne e-hemidiaph agm p epa a ions we e
moun ed in Pe spex chambe s o 3 ml capaci y h ough
which solu ions flowed. A e a 30-min equilib a ion
pe iod, he pe usion was s opped and he ne e end-
ings we e labeled o 40 min wi h 1 ␮M[
3
H]-choline
(specific ac i i y 2.5 ␮Ci nmol
⫺1
) unde elec ical s im-
ula ion a 1 Hz equency. A e he end o he labeling
pe iod he p epa a ions we e again supe used (15 ml
min
⫺1
) and he ne e s imula ion s opped. F om his
ime onwa ds, hemicholinium-3 (10 ␮M) was p esen o
p e en up ake o choline. A e a 60-min pe iod o
washou he pe usion was s opped. Ba h samples (2
ml) we e au oma ically collec ed e e y 3 min by emp-
ying and efilling he o gan ba h wi h he solu ion in
use, using a ac ion collec o (Gilson, FC 203B) cou-
pled o a pe is al ic pump (Gilson, Minipuls3) p o-
g ammed de ice. Aliquo s (0.5 ml) o he incuba ion
medium we e added o 3.5 ml o Packa d Ins a Gel II
(Me iden, CT, USA) scin illa ion cock ail. T i ium con-
en o he samples was measu ed by liquid scin illa-
ion spec ome y (% coun ing e ficiency: 40 ⫾2%)
a e app op ia e backg ound sub ac ion, which did
no exceed 5% o samples i ium con en . The adio-
ac i i y was exp essed as DPM g
⫺1
o we weigh o he
issue de e mined a he end o he expe imen . A e
he loading and washou pe iods, he p epa a ion con-
ained 5,542 ⫾248 ⫻10
3
DPM g
⫺1
and he es ing
elease was 132 ⫾12 ⫻10
3
DPM g
⫺1
in 3 min (n⫽8).
When he ac ional elease was calcula ed, his alue
p o ed o be 2.38 ⫾0.14% o he adioac i i y p esen
in he issue a he fi s collec ed sample. [
3
H]-ACh
elease was e oked s imula ing he ph enic ne e wi h
b ie high- equency ains (50 Hz o 5 sec, 40 ␮s pulse
wid h). Two s imula ion pe iods we e used: a 12 min
(S
1
) and a 39 min (S
2
) a e he end o washou (ze o
ime). Elec ical s imula ion o he ph enic ne e in-
c eased he elease o [
3
H]-ACh in a Ca
2⫹
- and e o-
do oxin-sensi i e manne (Co eia-de-Sa´ e al., 2000),
while he ou pu o [
3
H]-choline emained unchanged
(Wessle and Kilbinge , 1986), hus indica ing ha
ACh comes mainly om esicle exocy osis om depo-
la ized ne e e minals. I is unlikely ha nonquan al
ACh elease (Ka z and Miledi, 1977) accoun o he
o al amoun o ACh eleased upon elec ical s imula-
ion o he ph enic ne e. This assump ion is based on
findings indica ing ha he spon aneously eleasable
neu onal pool o ACh is no labeled wi h [
3
H]-choline
no is i eleased by elec ical ne e s imula ion (Mo-
lenaa e al., 1987), and i is comple ely exhaus ed
(wi hin minu es) in he p esence o hemicholinium-3
␣3␤2 NICOTINIC RECEPTORS ON MOTONEURONS 79
(Nikolsky e al., 1991). The e o e, e oked [
3
H]-ACh
elease was calcula ed by sub ac ing he basal i ium
ou flow om he o al i ium ou flow du ing each
s imula ion pe iod (Co eia-de-Sa´ e al., 1991). Tes
d ugs we e added 15 min be o e S
2
. In some expe i-
men s, incuba ion ime wi h BTX was p olonged o 45
min and S
2
was deli e ed a he 69
h
min a e he end
o washou . D ug e ec s we e exp essed by he a ios
S
2
/S
1
, i.e., he a io be ween he e oked [
3
H]-ACh e-
lease du ing he second s imula ion pe iod (in he p es-
ence o he es d ug) and he e oked [
3
H]-ACh elease
du ing he fi s s imula ion pe iod (wi hou he es
d ug). Pe cen age alues shown in figu es co espond
o pe cen age changes in S
2
/S
1
a ios as compa ed wi h
he S
2
/S
1
a io in con ol expe imen s (0.83 ⫾0.06, n⫽
6); ze o pe cen ep esen s iden i y be ween a ios.
Posi i e and nega i e alues ep esen acili a ion and
inhibi ion o e oked [
3
H]-ACh elease, espec i ely.
None o he d ugs used significan ly (P⬎0.05) changed
basal i ium ou flow.
Ma e ials and solu ions
Chemicals used we e: ␣-Bunga o oxin (BTX), choline
chlo ide, dihyd o-␤-e y h oidine hyd ob omide (DH-␤-
E), hemicholinium-3, hexame honium b omide (HEX),
mecamylamine (Meca), me hyllycaconi ine ci a e
(MLA), pi enzepine dihyd ochlo ide, d- ubocu a ine
chlo ide (d-TC) (Sigma, S . Louis, MO, USA); ␣-cono-
oxin MII (CTX MII) (Toc is Cookson, UK); ␮-cono oxin
GIIIB (CTX GIIIB), musca inic oxin 7 (Pep ide Ins i-
u e, Japan); [me hyl-
3
H]-choline chlo ide (e hanol so-
lu ion, 80 Ci mmol
⫺1
) (Ame sham, UK). Aqueous s ock
solu ions we e s o ed as ozen aliquo s a ⫺20°C. Di-
lu ions o hese s ock solu ions we e made daily and
app op ia e con ols we e done. The pH o he supe -
usion solu ion did no change by he addi ion o d ugs
in he maximum concen a ions applied o he p epa-
a ions.
S a is ics
The da a a e exp essed as mean ⫾SE, om nexpe -
imen s. S a is ical significance o expe imen al esul s
was analyzed by one-way analysis o a iance
(ANOVA) ollowed by Dunne ’s modified - es . P⬍
0.05 was conside ed o ep esen significan di e ence.
RESULTS
Blockade o muscle- ype nico inic ecep o s
con aining ␣1-subuni s educe e anic ension
Changes in he ampli ude o e anic peak ension
p oduced by nico inic ecep o an agonis s was aken
as a measu e o he pos junc ional ac i i y o hese
compounds. Figu e 1 shows ha BTX (0.003–0.1 ␮M),
d-TC (0.1–0.7 ␮M), Meca (0.1–300 ␮M), and HEX (10–
3,000 ␮M) dec eased e anic peak ension in a concen-
a ion-dependen manne . Failu e o de ec ne e-
e oked diaph agm con ac ions in esponse o e anic
ains (50 Hz o 5 sec) was obse ed wi h BTX (0.1
␮M), d-TC (0.7 ␮M), Meca (300 ␮M), and HEX (3,000
␮M). As BTX exhibi s slow binding kine ics and i s
ac ion is essen ially i e e sible, we pe o med expe i-
men s whe e he p eincuba ion ime was p olonged
om 12 o 45 min. Reduc ion o e anic peak ension
ollowing a 45-min con ac wi h BTX (0.003–0.1 ␮M)
was no s a is ically di e en (P⬎0.05) om ha
ob ained using a 12-min incuba ion pe iod (da a no
shown). Dep ession o e anic peak ension wi h DH-
␤-E (0.03–10 ␮M), which blocks p e e en ially ␣4␤2-
and ␣3␤2-con aining ecep o s, MLA (0.003–3 ␮M), a
p e e en ial ␣7- ecep o an agonis , and CTX MII
(0.001–0.3 ␮M), a selec i e ␣3␤2- ecep o an agonis
ha also blocks ecep o s con aining he ␣6 subuni ,
did no exceed 30%. Thus, educ ion in e anic peak
ension has an an agonis p ofile wi h a ank o de o
po ency o BTX ⬎d-TC ⰇMeca ⬎HEX. This is in
ag eemen wi h p e ious s udies sugges ing ha nico-
inic ecep o s localized on skele al muscle con ain ␣1-
subuni s (Schue ze and Role, 1987; Salpe e e al.,
1988).
nAChR unde lying e anic ade possess a
dis inc an agonis p o ile om he
muscula ecep o ype
Figu e 2A shows pen- eco de aces o ne e-e oked
muscle con ac ions ob ained du ing sho high- e-
quency ains (50 Hz o 5 sec). In con ol condi ions, a
Fig. 1. Concen a ion– esponse cu es o he e ec s o nico inic
ecep o an agonis s on he ampli ude o ne e-induced e anic peak
ension. Te anic esponses we e elici ed once e e y 15 min by s imu-
la ing he ph enic ne e wi h b ie high- equency ains (50 Hz o 5
sec, 40 ␮s pulse wid h). d-Tubocu a ine (d-TC), hexame honium
(HEX), mecamylamine (Meca), dihyd o-␤-e y h oidine (DH-␤-E),
me hyllycaconi ine (MLA), ␣-bunga o oxin (BTX), and ␣-cono oxin
MII (CTX MII) we e applied in a cumula i e manne and con ac ed
he p epa a ion a leas 12 min be o e eco dings. The o dina es a e
pe cen age o maximal e anic peak ension (100%) obse ed in con-
ol condi ions (in he absence o nico inic an agonis s). The e ical
ba s ep esen ⫾SE o 3–6 expe imen s ( o each cu e) and a e
shown when hey exceed he symbols in size.
80 M. FARIA ET AL.
b ie acili a ion (b ⬎a) was e iden du ing he cou se
o e ani (50 Hz o 5 sec), i.e., muscle ension was
ansien ly inc eased when high- equency epe i i e
pulses we e deli e ed o he ne e. Inc easing concen-
a ions o d-TC (0.1–0.7 ␮M, Fig. 2A), Meca (0.1–300
␮M), and HEX (10–3,000 ␮M) caused a e y in ense
ade (b ⬍a) o e anic con ac ions (Fig. 3) in pa allel
wi h a significan educ ion o he maximal e anic
ension (c . Fig. 1). Nei he BTX (0.003–0.1 ␮M) no
MLA (0.003–3 ␮M) p oduced e anic ade (Fig. 3), al-
bei BTX (0.003–0.1 ␮M) s ongly dep essed he max-
imal e anic ension (Figs. 1, 2A).
To in es iga e he possibili y o a di ec ac ion o he
nico inic an agonis s on muscle con ac ile p ope ies,
we s udied he e ec o d-TC on e anic ension induced
by di ec muscle s imula ion. When applied in concen-
a ions abo e hose ha caused comple e neu omus-
cula block, d-TC (1 and 5 ␮M) and BTX (0.3 ␮M) we e
i ually de oid o e ec on muscle ension induced by
e anic field s imula ion (50 Hz o 5 sec) (Fig. 2B). In
addi ion, measu emen s o e anic ension and ading
may depend on he h eshold o ac i a ion o muscle
ac ion po en ial, which would be misin e p e ed as be-
ing due o an indi ec p esynap ic e ec . To e alua e
his possibili y we compa ed he dep ession o con ac-
ile esponses caused by he nico inic ecep o an ago-
nis s wi h he e ec o ␮-cono oxin GIIIB (CTX GIIIB),
a sodium channel blocke in skele al muscle wi h no
ac ion on channels p esen in axons and mo o ne e
e minals (C uz e al., 1985). CTX GIIIB (0.01–0.6 ␮M)
dec eased e anic peak ension in a concen a ion-de-
penden manne wi h no ob ious ading phenomena
being associa ed (Fig. 2). Muscula pa alysis was ob-
ained wi h CTX GIIIB (0.6 ␮M) when e anic ains
(50 Hz o 5 sec) we e deli e ed ei he indi ec ly, o he
ph enic ne e unk, o di ec ly, o he muscle fibe s.
Da a om di ec muscle s imula ion p o ocols and he
absence o e anic ading in he p esence o CTX GIIIB
clea ly con as wi h he esul s ob ained wi h d-TC
(see Fig. 2), u he indica ing ha a p esynap ic ac ion
migh be in ol ed.
Neu onal ␣3␤2 nAChR block educes [
3
H]-ACh
elease igge ed by high- equency ains
To add ess he ole o neu omuscula blocking
agen s on p ejunc ional nico inic ecep o s, we com-
pa ed hei abili y o p oduce e anic ade and o de-
c ease [
3
H]-ACh elease e oked by high- equency
s imula ion ains (50 Hz o 5 sec). Figu e 4A illus-
a es he ime cou se o i ium ou flow in expe i-
men s whe e CTX MII (0.1 ␮M), d-TC (1 ␮M), and BTX
(0.3 ␮M) we e applied 15 min be o e S
2
. As can be seen
om hese ypical expe imen s, e oked [
3
H]-ACh e-
lease was dec eased in he p esence o CTX MII (0.1
Fig. 2. Compa ison be ween he e ec s o wo nico inic ecep o
an agonis s (d- ubocu a ine and ␣-bunga o oxin) and ␮-cono oxin
GIIIB, a sodium channel blocke in skele al muscle, on e anic muscle
ension induced by high- equency (50 Hz o 5 sec) s imula ion ains
deli e ed o he ph enic ne e (indi ec s imula ion, A) o o dia-
ph agm muscle fibe s (di ec s imula ion, B). A: Typical eco ding
aces o ne e-e oked hemidiaph agm con ac ions ob ained du ing
b ie e anic ains (50 Hz o 5 sec), in he absence (C ) and in he
p esence o d- ubocu a ine (d-TC, 0.3 and 0.7 ␮M), ␣-bunga o oxin
(BTX, 0.03 and 0.1 ␮M), and ␮-cono oxin GIIIB (CTX GIIIB, 0.3 and
0.6 ␮M). The small ho izon al lines indica e he du a ion o e anic
s imula ion (5 sec); e ical calib a ion: 50 mN. B: No e ha d-TC (1
o 5␮M) and BTX (0.3 ␮M), applied in concen a ions abo e hose
equi ed o cause comple e neu omuscula block we e i ually de oid
o e ec s on he con ac ile esponses induced by di ec muscle s im-
ula ion. In con as , CTX GIIIB (0.6 ␮M) abolished con ac ions
caused by di ec muscle depola iza ion.
Fig. 3. E ec s o nico inic ecep o an agonis s on e anic ading
induced by high- equency (50 Hz o 5 sec) s imula ion ains deli -
e ed o he ph enic ne e. Te anic ade was calcula ed as he a io (R)
be ween he ensions eco ded a he end (b) and a he beginning (a)
o he e anic esponse (R ⫽b/a) (see Fig. 2). d-Tubocu a ine (d-TC),
hexame honium (HEX), mecamylamine (Meca), dihyd o-␤-e y h oi-
dine (DH-␤-E), me hyllycaconi ine (MLA), ␣-bunga o oxin (BTX), and
␣-cono oxin MII (CTX MII) we e applied in a cumula i e manne and
con ac ed he p epa a ion a leas 12 min be o e eco dings. On he
o dina e, a io (R) is exp essed as a pe cen age o ha ob ained in
con ol (C ) condi ions (in he absence o nico inic an agonis s), aken
as 100%. The e ical ba s ep esen ⫾SE o 3–6 expe imen s ( o
each cu e) and a e shown when hey exceed he symbols in size.
␣3␤2 NICOTINIC RECEPTORS ON MOTONEURONS 81

␮M) and d-TC (1 ␮M), bu no when BTX (0.3 ␮M) was
added. The an agonis ank po ency o de o inhibi (by
abou 50–70%) he elease o [
3
H]-ACh (50 Hz o 5 sec)
was CTX MII (0.1 ␮M) ⬎DH-␤-E (1 ␮M) ⬃d-TC (1
␮M) ⬎Meca (100 ␮M) ⬎HEX (1000 ␮M) (Fig. 4B). The
lack o BTX e ec a he p ejunc ional le el was ob-
se ed e en when he ime o incuba ion was p olonged
o 45 min and i was applied in a concen a ion (0.3
␮M) abo e ha necessa y o cause comple e muscula
pa alysis (da a no shown). I is also wo h no ing
ha applica ion o BTX did no significan ly (P⬎
0.05) a ec he basal i ium ou flow (Fig. 4A; bu see
e.g., Apel e al., 1995). This ully ag ees wi h da a
showing ha ailu e o BTX (0.003–0.1 ␮M) o in-
duce p esynap ic undown o e anic con ac ions
can be dissocia ed om i s abili y o dec ease e anic
peak ension (see abo e). Thus, he p esen esul s
sugges ha au o acili a ion o ACh elease om
mo o ne e e minals is p obably media ed by ␣3␤2-
con aining nico inic ecep o s sensi i e o blockade
by CTX MII.
Reducing he sa e y ac o o neu omuscula
ansmission signi ican ly po en ia es e anic
ade caused by neu onal nico inic
ecep o an agonis s
In gene al, he amoun o ansmi e eleased pe
ne e impulse is g ea e han ha equi ed o igge
an ac ion po en ial in he muscle fibe , al hough he
ansmission sa e y ma gin may become c i ical in
pa hological condi ions (e.g., myas henic synd omes).
Because o he high sa e y ac o o neu omuscula
ansmission, dep ession o ne e-e oked muscle con-
ac ions due o p ejunc ional ac ing d ugs migh no
always eflec he magni ude o ansmi e elease
inhibi ion (see o a e iew, see Wood and Sla e , 2001).
This migh explain why nico inic blocking agen s like
CTX MII (0.1 ␮M) and DH-␤-E (1 ␮M) a e mo e po en
o inhibi (⫺56 ⫾9%, n⫽5 and ⫺50 ⫾6%, n⫽4,
espec i ely) e oked [
3
H]-ACh elease (Fig. 4) han o
cause dep ession o e anic con ac ions (Figs. 5, 6).
Inc easing magnesium concen a ion in he ba hing
fluid is a use ul s a egy o dec ease he sa e y ac o o
Fig. 4. E ec s o nico inic ecep o an agonis s on [
3
H]-ACh e-
lease om he a mo o ne e endings e oked by high- equency (50
Hz o 5 sec) ains. A: Time cou se o i ium ou flow om a ph enic
ne e e minals in he absence (Con ol, F) and in he p esence (Œ)o
␣-bunga o oxin (BTX, 0.3 ␮M), d- ubocu a ine (d-TC, 1 ␮M), and
␣-cono oxin MII (CTX MII, 0.1 ␮M). A e he labeling and washou
pe iods (ze o ime), [
3
H]-ACh elease was elici ed by s imula ing he
ph enic ne e wice (S
1
a he 12 h min and S
2
a he 39 h min) a a
equency o 50 Hz o 5 sec. T i ium ou flow was measu ed in samples
collec ed e e y 3 min. The nico inic an agonis s we e applied a leas
12 min be o e S
2
(as indica ed by he ho izon al ba s). No e ha
spon aneous i ium ou flow was no significan ly modified in he
p esence o he d ugs. B: Inhibi o y e ec s o ␣-cono oxin MII (CTX
MII, 0.1 ␮M), ␣-bunga o oxin (BTX, 0.3 ␮M), dihyd o-␤-e y h oidine
(DH-␤-E, 1 ␮M), d- ubocu a ine (d-TC, 1 ␮M), mecamylamine (Meca,
100 ␮M), and hexame honium (HEX, 1000 ␮M) on e oked [
3
H]-ACh
elease om mo o ne e e minals de e mined as desc ibed in (A).
The o dina es a e pe cen age inhibi ion o [
3
H]-ACh elease as com-
pa ed o con ol, in he absence o added d ugs. Each column ep e-
sen s pooled da a om 4–11 expe imen s. The e ical ba s ep esen
⫾SE. *P⬍0.05 (one-way ANOVA ollowed by Dunne ’s modified
- es ) when compa ed wi h ze o pe cen .
82 M. FARIA ET AL.
synap ic ansmission by educing he amoun o ans-
mi e being eleased pe s imula ion pulse (del
Cas illo and Ka z, 1954; Pa on and Waud, 1967). We
epo ed p e iously ha inc emen s o he magnesium
con en in he bu e (up o 8.5 mM) dec eased he
e oked [
3
H]-ACh elease by 53 ⫾9% (n⫽4), wi hou
a ec ing agonis -induced p esynap ic nico inic acili a-
ion (Co eia-de-Sa´ and Ribei o, 1994). To p obe o he
appa en disc epancy exis ing be ween he magni ude
o ansmi e elease inhibi ion and he ading phe-
nomena, we ee alua ed he e ec s o DH-␤-E (0.03–10
␮M) and CTX MII (0.001–0.3 ␮M) in condi ions whe e
he sa e y ma gin o neu omuscula ansmission was
pa ially educed wi h high magnesium concen a ions
(6–7 mM). Figu e 5 illus a es pen- eco de aces
aken om ep esen a i e expe imen s wi h DH-␤-E (3
and 10 ␮M) and CTX MII (0.1 and 0.3 ␮M), whe e
ne e-induced e anic peak ension was educed by
abou 50% using 6–7 mM MgCl
2
. The ampli ude o
aces ob ained wi h no mal (1 mM) and high magne-
sium concen a ions we e no malized o acili a e com-
pa ison (see calib a ion ba in he figu e). Dep essions
o e anic peak ension due o bo h DH-␤-E (0.03–10
␮M) and CTX MII (0.001–0.3 ␮M) we e significan ly
(P⬍0.05) po en ia ed upon inc easing magnesium
con en in he bu e (Fig. 6). A comple e neu omuscu-
la block was ob ained wi h 0.3 and 10 ␮M concen a-
ions o DH-␤-E in he p esence o 7 and 6 mM MgCl
2
,
espec i ely.
I is wo h no ing ha magnesium (6–7 mM) sligh ly
enhanced e anic acili a ion (see R alues in Figs. 6B,
D). This is a well-known phenomenon seen du ing
high- equency epe i i e ne e s imula ion when
quan al ou pu is low (see e.g., an de Kloo and
Molgo´, 1994). Albei , p ese a ion o he e anic ascen-
dan in high magnesium solu ions, simul aneous appli-
ca ion o DH-␤-E (0.03–10 ␮M) significan ly (P⬍0.05)
enhanced ading (Fig. 6B). Tension a he end o e ani
was i ually abolished when 1 and 10 ␮M DH-␤-E was
added in he p esence o 7 and 6 mM MgCl
2
, espec-
i ely. Unde low quan al condi ions, CTX MII (0.001–
0.3 ␮M) significan ly (P⬍0.05) a enua ed e anic
acili a ion in a concen a ion-dependen manne (Fig.
6D). CTX MII (0.3 ␮M, n⫽3) dec eased he R a io
(b/a) om 1.50 ⫾0.02 (n⫽3, no mal Ty ode bu e ) o
1.20 ⫾0.05 (n⫽3) when 7 mM MgCl
2
was added o he
incuba ion media.
d-TC (0.7 ␮M), Meca (300 ␮M), and HEX (3,000 ␮M)
used in concen a ions high enough o dec ease he
sa e y ac o o neu omuscula ansmission induced
he undown o e anic con ac ions un il comple e
neu omuscula block (see Fig. 3). These achie emen s
did no equi e he aising o magnesium concen a-
ion, because hese agen s may simul aneously block
p e- and pos junc ional nico inic si es.
Blockade o musca inic M
1
au o ecep o s
educe ansmi e elease wi hou
p oducing e anic ade
In addi ion o he sho - e m nico inic posi i e eed-
back mechanism, ACh may inc ease i s own elease by
ac ing a musca inic M
1
ecep o s on mo o ne e e -
minals (see e.g., Wessle , 1989; Oli ei a e al., 2002).
As illus a ed in Figu e 7A, he musca inic M
1
ecep o
an agonis pi enzepine (10 nM) inhibi ed (26 ⫾5%, n ⫽
4) he elease o [
3
H]-ACh induced by 50 Hz equency
ains. This inhibi o y ac ion was mimicked (37 ⫾6%,
n⫽5) by he musca inic oxin 7 (MT-7, 1 nM, da a no
shown) isola ed om he enom o he g een mamba
(Dend oaspis angus iceps), which exhibi s high sub-
ype selec i i y o M
1
(pK
B
⬃9.8) ecep o s (Adem and
Ka lsson, 1997).
Pi enzepine (1–30 nM), applied cumula i ely a leas
12 min be o e eco dings, dec eased e anic peak en-
sion in a concen a ion-dependen manne . Dep ession
o e anic con ac ions due o pi enzepine (1–30 nM)
did no exceed 20% in no mal Ty ode solu ion (MgCl
2
,
1 mM) (Fig. 7B), bu i was significan ly (P⬍0.05)
Fig. 5. Rep esen a i e eco dings o ne e-e oked hemidiaph agm
e ani (50 Hz o 5 sec) in he p esence o dihyd o-␤-e y h oidine
(DH-␤-E, 3 and 10 ␮M) and ␣-cono oxin MII (CTX MII, 0.1 and 0.3
␮M): influence o he sa e y ma gin o neu omuscula ansmission.
Reco dings in Aand C(no mal quan al ou pu ) we e ob ained in
no mal Ty ode bu e (MgCl
2
, 1 mM), and hose in Band D(low
quan al ou pu ) we e ob ained in high magnesium condi ions (MgCl
2
,
6–7 mM). DH-␤-E (3 and 10 ␮M) and CTX MII (0.1 and 0.3 ␮M) we e
applied in a cumula i e manne o pe iods o 12 min be o e each 50
Hz equency s imula ion ain. The small ho izon al line indica es
he du a ion o e anic s imula ion (5 sec). Please no e ha he am-
pli ude o aces ob ained in no mal (Aand C, e ical calib a ion: 50
mN) and high (Band D, e ical calib a ion: 25 mN) magnesium
condi ions we e no malized o acili a e compa isons.
␣3␤2 NICOTINIC RECEPTORS ON MOTONEURONS 83
po en ia ed (46 ⫾7%, n⫽3) when he magnesium
con en in he bu e was aised o 6 mM (Fig. 7C). In
con as o he findings ob ained wi h se e al nico inic
channel blocke s (e.g., DH-␤-E, CTX MII, d-TC), pi en-
zepine (30 nM) was i ually de oid o e ec on e anic
acili a ion e en a e dec easing he sa e y ac o o
neu omuscula ansmission. Unde hese condi ions,
pe cen a ia ion o R a io (b/a) was no highe han
3–7% when compa ed o con ols ob ained in no mal
Ty ode’s solu ion (1.23 ⫾0.07, n⫽3) o a e aising
magnesium le els (1.35 ⫾0.06, n⫽3).
DISCUSSION
In his s udy we demons a e ha he a neu omus-
cula junc ion is equipped wi h ␣3␤2-con aining neu-
onal nAChRs media ing acili a ion o ACh elease, in
addi ion o he classical muscle- ype nAChR con aining
he ␣1 subuni . Sub ype-specific nico inic an agonis s
(e.g., BTX, DH-␤-E, CTX MII) had dis inc p ofiles o
inhibi e oked [
3
H]-ACh elease and o dep ess e anic
peak ension, clea ly indica ing ha p e- and pos junc-
ional ecep o s ha e di e en pha macological p op-
e ies. Unlike e anic peak dep ession due o he “pu e”
muscula elaxing agen , BTX, ading o e anic con-
ac ions induced by DH-␤-E and CTX MII consis ed
p ima ily o he inhibi ion o ACh elease om mo o
ne e e minals. This p o ides u he suppo o he
hypo hesis ha e anic ade is due o an unde lying
a enua ion o nico inic au o acili a ion a he han o
a use-dependen block o pos junc ional nico inic ecep-
o s (Wilson and Nicholson, 1997). Due o he high
sa e y ma gin o neu omuscula ansmission, epe -
cussions o he fine- uning nico inic modula ion o
ansmi e elease a he pos junc ional le el equi e
significan dec eases in he synap ic quan al con en ,
like hose obse ed du ing high- equency ains o
a e inc easing he magnesium con en in he bu e .
In con as o he pi o al ole o nAChRs in au o-
nomic neu o ansmission and o ini ia e muscle con-
ac ion, neu onal nAChRs a e conside ed o exe a
modula o y influence (Wonnaco , 1997). In na i e neu-
onal sys ems, knowledge o he subuni composi ion o
nAChRs is gene ally lacking and only a ew majo
sub ypes ha e been iden ified. These include ␣4␤2*
nAChR, which is ela i ely abundan in he CNS
(Flo es e al., 1996). The o he majo sub ype is com-
Fig. 6. E ec s o dihyd o-␤-e y h oidine (DH-␤-E, 0.03–10 ␮M)
and ␣-cono oxin MII (CTX MII, 0.001–0.3 ␮M) on e anic peak ension
and ade (R ⫽b/a) in condi ions whe e he sa e y ac o o neu omus-
cula ansmission was educed. Te anic esponses we e induced once
e e y 15 min by s imula ing he ph enic ne e wi h b ie high- e-
quency ains (50 Hz o 5 sec, 40 ␮s pulse wid h). DH-␤-E (0.03–10
␮M, Aand B) and CTX MII (0.001–0.3 ␮M, Cand D) we e applied in
a cumula i e manne and con ac ed he p epa a ion a leas 12 min
be o e eco dings. A,C: The o dina es a e pe cen age o maximal
e anic peak ension (100%) de e mined in he absence o nico inic
an agonis s. B,D: The a io (R) is exp essed as a pe cen age o ha
ob ained in he absence o nico inic an agonis s, aken as 100%. R
alues o each se o expe imen s a e indica ed o compa ison. The
e ical ba s ep esen ⫾SE o nexpe imen s and a e shown when
hey exceed he symbols in size. *P⬍0.05 (one-way ANOVA ollowed
by Dunne ’s modified - es ) as compa ed wi h he e ec o each
nico inic ecep o an agonis in no mal Ty ode bu e (MgCl
2
1 mM).
84 M. FARIA ET AL.
p ised o ␣7 subuni s, which o m homome ic ecep o s
in bo h cen al and pe iphe al ne ous sys ems (Chen
and Pa ick, 1997). A a ie y o he e ome ic ␣3*
nAChRs may also exis in he pe iphe al ne ous sys-
em; he p edominan sub ype con ains ␣3 and ␤4 sub-
uni s bu may also assemble wi h ␣5 and/o ␤2 sub-
uni s (Con oy and Be g, 1995; Flo es e al., 1996).
A emp s o iden i y he subuni composi ion o
nAChRs p esen a mo o ne e e minals ha e been
made using nico inic an agonis s lacking sub ype se-
lec i i y (e.g., Vizi e al., 1995). In his a icle we show
ha he an agonis ank po ency o de o p oduce neu-
omuscula e anic ade and o educe he elease o
[
3
H]-ACh e oked by high- equency ains was CTX
MII ⬎DH-␤-E ⬃d-TC ⬎Meca ⬎HEX. Nei he BTX
no MLA caused e anic ade and/o modified he e-
lease o [
3
H]-ACh, i ually excluding he in ol emen
o neu onal BTX-sensi i e ecep o s (like ␣7*, ␣8*, and
␣9*) in he nico inic posi i e eedback mechanism. O h-
e s ha e ound ha BTX p oduced a disp opo iona ely
highe educ ion on he ampli ude o minia u e end-
pla e po en ials when compa ed o he ampli ude o
endpla e po en ials eco ded du ing b ie e anic ains
(Dome e al., 1995). Howe e , one canno exclude he
possibili y ha his ansien acili a ion o ACh e-
lease p io o he es ablishmen o he neu omuscula
block could be due o phospholipase A
2
ac i i y (Fa hi
e al., 2001), which is a known con aminan o se e al
comme cially a ailable snake oxins, including BTX
(see e.g., Apel e al., 1995). Equipo ency be ween DH-
␤-E and d-TC o inhibi e oked [
3
H]-ACh elease, o-
ge he wi h he highe po ency o hese agen s as com-
pa ed o Meca, make he in ol emen o ␣4*- and ␣3␤4-
con aining ecep o s also highly imp obable (see e.g.,
Dwoskin and C ooks, 2001). Since, DH-␤-E is a ela-
i ely weak an agonis a ␣3␤4- (ganglionic-like) and
(␣1)
2
ε␤1␦- (muscle- ype) ecep o s as compa ed o he
␣3␤2 sub ype (Cha ez-No iega e al., 1997) and e oked
[
3
H]-ACh elease was highly sensi i e o CTX MII (a
p e e en ial ␣3␤2-an agonis a he nanomola concen-
a ion ange) (Ca ie e al., 1996), ou da a indica e
ha acili a o y nAChRs loca ed a he a mo o ne e
e minals exhibi a ␣3␤2 subuni configu a ion. Immu-
nohis ochemical s udies pe o med in mouse dia-
ph agms suppo he iew ha ␣3-con aining nAChRs
may exis a he p ejunc ional le el (Tsuneki e al.,
1995). The in ol emen o CTX MII-sensi i e neu onal
␣6-he e ocep o s (Ku ya o e al., 2000) canno be ex-
cluded om he p esen da a, despi e he ac ha in
he chick e ina hese ecep o s a e also blocked by
nanomola concen a ions o MLA (Vaila i e al., 1999).
The an agonis p ofile ound in he elease expe i-
men s clea ly di e s om ha conce ning dep ession
o e anic peak ension (BTX ⬎d-TC ⰇMeca ⬎HEX),
which is a well-known phenomenon esul ing om he
blockade o muscle- ype ␣1-con aining nAChRs
(Schue ze and Role, 1987; Salpe e e al., 1988). I is
no su p ising ha d-TC, Meca, and HEX could simul-
aneously dep ess e anic peak ension, educe e oked
[
3
H]-ACh elease, and induce e anic ade, as nonselec-
i e agen s can an agonize coope a i ely bo h neu onal
and muscula nAChRs (see e.g., de Oli ei a and Ol-
i ei a, 1999). In con as , ansmi e elease inhibi-
ion caused by neu onal nAChR an agonis s, like DH-
␤-E- and CTX MII, was associa ed wi h a mild ading
phenomenon. This appa en disc epancy was a enu-
a ed once he sa e y ac o o neu omuscula ansmis-
sion was dec eased by educing he p obabili y o
ansmi e elease using high magnesium concen a-
ions in he bu e (del Cas illo and Ka z, 1954; Pa on
and Waud, 1967; see also Co eia-de-Sa´ and Ribei o,
Fig. 7. E ec o he musca inic M
1
- ecep o an agonis , pi enz-
epine, on ansmi e elease and muscula ension induced by high-
equency (50 Hz o 5 sec) s imula ion ains deli e ed o he ph enic
ne e. A: Time cou se o i ium ou flow om a ph enic ne e
e minals in he absence (Con ol, F) and in he p esence (Œ)o
pi enzepine (10 nM), applied 15 min be o e S
2
(as indica ed by he
ho izon al ba ). A e he labeling and washou pe iods (ze o ime),
[
3
H]-ACh elease was elici ed by s imula ing he ph enic ne e wice
(S
1
a he 12 h min and S
2
a he 39 h min) a a equency o 50 Hz
du ing 5 sec. T i ium ou flow was measu ed in samples collec ed
e e y 3 min. B,C: Typical eco ding aces o ne e-e oked hemidia-
ph agm con ac ions obse ed du ing b ie e anic ains (50 Hz o 5
sec), in he absence (C ) and in he p esence o pi enzepine (10 and 30
nM), ob ained in no mal Ty ode bu e (B, MgCl
2
, 1 mM) and in high
magnesium condi ions (C, MgCl
2
, 6 mM). Pi enzepine (1–30 nM) was
applied in a cumula i e manne o pe iods o 12 min be o e e ani.
The small ho izon al line indica es he du a ion o e anic s imula ion
(5 sec). Please no e ha he ampli ude o aces ob ained in no mal (B,
e ical calib a ion: 50 mN) and high (C, e ical calib a ion: 25 mN)
magnesium condi ions we e no malized o acili a e compa isons.
␣3␤2 NICOTINIC RECEPTORS ON MOTONEURONS 85