PERSPECTIVE
published: 24 May 2016
doi: 10.3389/ nsyn.2016.00012
The Ac i e and Pe iac i e Zone
O ganiza ion and he Func ional
P ope ies o Small and La ge
Synapses
Raquel Cano and Lucia Taba es*
Depa men o Medical Physiology and Biophysics, School o Medicine, Uni e si y o Se ille, Se ille, Spain
Edi ed by:
Ma ín Camma o a,
Fede al Uni e si y o Rio G ande do
No e, B azil
Re iewed by:
Je ey Sco Diamond,
Na ional Ins i u es o Heal h, USA
Michele H. Jacob,
Tu s Uni e si y, USA
*Co espondence:
Lucia Taba es
[email p o ec ed]
Recei ed: 21 Ma ch 2016
Accep ed: 09 May 2016
Published: 24 May 2016
Ci a ion:
Cano R and Taba es L (2016) The
Ac i e and Pe iac i e Zone
O ganiza ion and he Func ional
P ope ies o Small and
La ge Synapses.
F on . Synap ic Neu osci. 8:12.
doi: 10.3389/ nsyn.2016.00012
The a i al o an ac ion po en ial (AP) a a synap ic e minal elici s highly synch onized
quan a elease. Repe i i e APs p oduce successi e synap ic esicle (SV) usions ha
equi e managemen o spen SV componen s in he p esynap ic memb ane wi h
minimum dis u bance o he sec e o y appa a us. To his end, he synap ic machine y
is s uc u ed acco dingly o he s eng h and he ange o equencies a which each
pa icula synapse ope a es. This esul s in a ia ions in he numbe and dimension
o Ac i e Zones (AZs), amoun and dis ibu ion o SVs, and p obably, in he p ima y
endocy ic mechanisms hey use. Unde s anding be e how hese s uc u al di e ences
de e mine he unc ional esponse in each case has been a ma e o long- e m
in e es . He e we e iew he s uc u al and unc ional p ope ies o h ee dis inc ypes
o synapses: he neu omuscula junc ion (NMJ; a gian , highly eliable synapse ha
mus exocy ose a la ge numbe o quan a wi h each s imulus o gua an ee exci a ion
o he pos synap ic cell), he hippocampal exci a o y small synapse (which mos o en
has a single elease si e and a ela i ely small pool o esicles), and he ce ebella
mossy ibe -g anule cell synapse (which possesses hund eds o elease si es and is
able o ansloca e, dock and p ime esicles a high speed). We will ocus on how he
elease appa a us is o ganized in each case, he ela i e amoun o esicula memb ane
ha needs o be accommoda ed wi hin he pe iAZ upon s imula ion, he di e en
mechanisms o e ie ing he excess o memb ane and inally, how hese ac o s may
in luence he unc ioning o he elease si es.
Keywo ds: ac i e zone, neu o ansmi e elease, endocy osis, pe iac i e zone, elease si es
ORGANIZATION OF THE RELEASE APPARATUS IN SMALL
AND LARGE SYNAPSES
Synap ic e minals di e in hei s eng h and sho - e m plas ici y, as well as in he size
and spa ial o ganiza ion o he sec e o y appa a us, mainly in he dimensions, shape, and
amoun o hei Ac i e Zones (AZs), and in he size o hei ecycling pools o synap ic
esicles (SVs; A wood and Ka unani hi, 2002; Zhai and Bellen, 2004). Mos e minals in
he cen al ne ous sys em (CNS) ha e a small numbe (1–8) o AZs, and he size o he
ecycling pool o SVs is no la ge. Fo ins ance, exci a o y ne e e minals in a ea CA1 o he
mouse hippocampus ha e a single AZ, abou 10 docked esicles pe AZ, and a ecycling pool o
F on ie s in Synap ic Neu oscience | www. on ie sin.o g 1May 2016 | Volume 8 | A icle 12
Cano and Taba es Small and La ge Synapse P ope ies
abou 200 SVs (Schiko ski and S e ens, 1997; Mu hy e al.,
2001; Rizzoli and Be z, 2005). La ge e minals, on he o he
hand, such he a ce ebella mossy ibe –g anule cell synapse,
he calyx o Held and he mouse neu omuscula junc ion (NMJ),
ha e hund eds o AZs and a la ge ecycling pool o SVs.
These s uc u al di e ences a e in acco dance wi h he speci ic
unc ional oles o each synapse ype. When he pos synap ic
cell esponse is de e mined by he spa ial and empo ally
in eg a ed ac i i y o hund eds o small e minals (Figu e 1A),
he numbe o quan a eleased pe impulse (quan um con en )
pe e minal is small. Con e sely, when he pos synap ic cell
ecei es in o ma ion om only a ew la ge ne e e minals,
he sensi i i y and ideli y o he ansmission a e usually e y
high. Fo example, in he ce ebella mossy ibe —g anule cell
synapse (Figu e 1B), a bu s o ac ion po en ials (APs) in a
single mossy ibe bou on could be su icien o gene a e spikes
a he g anule cell (Rancz e al., 2007). In his synapse, a
la ge ecycling pool o esicles also con ibu es o sus aining
ansmission a high equency (Sa iane and Sil e , 2006; Rancz
e al., 2007). Finally, when he pos synap ic cell is e y la ge
and ecei es only one inpu , he size o he p esynap ic e minal
is also big, as a e he numbe o AZs and he ecycling
pool o SVs. Typical examples a e he NMJ (Figu e 1C) and
he calyx o Held.
In e es ingly, e en indi idual neu ons can exhibi la ge
di e ences be ween neighbo ing synapses. The hippocampal
den a e g anule cell mossy ibe , o example, has 11–18 ela i ely
la ge bou ons, each wi h ens o AZs, and in addi ion small
e minals a ising om ilopodial ex ensions o he la ge ones
(Nicoll and Schmi z, 2005).
MEMBRANE LOAD DURING SYNAPTIC
ACTIVITY
Du ing synap ic ac i i y, esicles used a elease si es a e
ansloca ed o he pe iAZ. This p oduces a memb ane load
in his compa men , he magni ude o which depends on
he du a ion and equency o he s imula ion; ne e heless,
i s ela i e impac a ies wi h he AZ o ganiza ion o each
e minal. Fo example, a he mouse NMJ (Figu e 1C) om
he le a o au is longus (LAL) muscle, a pu e as muscle,
a hal -second s imulus ain o 50 APs eleases abou 1700
quan a, he size o eadily eleasable pool (RRP) o SVs in his
e minal (Ruiz e al., 2011). Assuming a mean SV diame e
o ∼40 nm, he o al memb ane load is ∼8.5 µm2(1700
SVs ×πd2). Howe e , gi en he small size o hei AZs
(0.0054 µm2(60 ×90 nm)); Fukunaga e al., 1983; Fukuoka e al.,
1987) and he dis ance be ween neighbo ing AZs) ∼0.5 µm;
Ruiz e al., 2011), he su ace a ea o each pe iAZ egion
(0.5 ×0.5 µm2−0.0054 µm2=0.24 µm2) inc eases only by
4.1% when he wo p ima y docked esicles wi hin each AZ
(Nagwaney e al., 2009) use.
In ano he la ge synapse, he ce ebella mossy ibe bou on,
which also has hund eds o elease si es (Figu e 1B), each one
hos ing ∼7–8 docked esicles (Xu-F iedman and Regeh , 2004),
he mean a ea o he AZ is abou ou old la ge han in mouse
mo o ne e e minals (0.0216 µm2), and he dis ance be ween
neighbo ing AZs is ∼0.5 µm (Xu-F iedman e al., 2001; Ruiz
e al., 2011). The e o e, in his cen al synapse, i all AZ docked
esicles a es use du ing phasic ne e ac i i y, he su ace a ea
o each pe iAZ (0.5 ×0.5 µm2−0.0216 µm2=0.2284 µm2)
inc eases by ∼16.5%.
In con as , in small cen al synapses (Figu e 1A), al hough
he quan al con en is much less, a simila numbe o
s imuli may p oduce a much la ge ela i e inc emen in
he p esynap ic memb ane su ace a ea. Fo example, in CA1
exci a o y hippocampal p esynap ic bou ons, which ha e a
mean p esynap ic su ace a ea o a ound 0.2 µm2(Schiko ski
and S e ens, 1997), and an AZ a ea o ∼0.027 µm2, i
10 SVs ( he mean size o he RRP) use wi h he p esynap ic
memb ane du ing 20 Hz, 2 s s imula ion, he su ace inc eases
by ∼0.05 µm2, which ep esen s a ∼29% inc ease o he pe iAZ
su ace a ea. The e o e, du ing high equency s imula ion, i he
excess o memb ane is no apidly emo ed om he AZ and/o
he pe iAZ (Roos and Kelly, 1999) by compensa o y endocy osis,
o ansloca ed o dis an egions o la e ission, he ela i e
accumula ion o esicula memb ane a he pe iAZ is la ge in
small synapses, mainly because hei g ea e numbe o eady-
o-go esicles pe AZ, and hei ela i ely smalle pe i-AZ a ea
(Figu e 1D).
WHEN DOES ENDOCYTOSIS START AND
HOW FAST DOES IT GO?
Endocy osis is a complex p ocess ha has been s udied mainly by
ul as uc u al analysis, elec ical capaci ance measu emen s, and
by eal- ime imaging o luo escen molecules associa ed wi h he
memb anes. These and o he echniques ha e p o ided e idence
ha slow and as modes o endocy osis exis (Figu e 2). Fo
example, single SV cla h in-media ed endocy osis is a ela i ely
slow p ocess, in he ange o ens o seconds (Heuse and
Reese, 1973; G anse h e al., 2006; Balaji e al., 2008; Clay on
e al., 2008). In he second mode o endocy osis, he so-called
kiss-and- un mode, a e he opening o he usion po e and
eleasing o he s o ed ma e ial, he esicle memb ane is apidly
eco e ed (∼1s;Cecca elli e al., 1972; Richa ds e al., 2000,
2005; A a anis e al., 2003; Gandhi and S e ens, 2003). Also,
he e ie al o a la ge pa ch o memb ane can be achie ed
a once by wha i is called bulk endocy osis (Heuse and
Reese, 1973; Mille and Heuse , 1984; Hol e al., 2003; Pailla
e al., 2003; Clay on e al., 2007; Wu and Wu, 2007; Hayashi
e al., 2008). Finally, an ul a as mode o endocy osis, only
ac i e a physiological empe a u e, has been desc ibed in which
memb ane pa ches, co esponding o he a ea o abou 4 SVs, a e
e ie ed wi hin 50–100 ms a e s imula ion (Wa anabe e al.,
2013a,b, 2014).
Despi e he in o ma ion p o ided by he di e en echniques,
he mode o esicle ecycling in each synapse ype is s ill
con o e sial. Fo ins ance, he kiss-and- un mode has been
desc ibed a he NMJ (Cecca elli e al., 1973), a he calyx o Held
(He e al., 2006), and a hippocampal neu ons in cul u e (Ha a a
e al., 2006). Ne e heless, in small hippocampal bou ons, many
au ho s ha e epo ed ha endocy osis s a s wi h a delay o a
ew seconds upon s imula ion and p oceeds slowly. Fo example,
F on ie s in Synap ic Neu oscience | www. on ie sin.o g 2May 2016 | Volume 8 | A icle 12
Cano and Taba es Small and La ge Synapse P ope ies
FIGURE 1 | S uc u al and unc ional p ope ies o di e en ne e e minals. (A) Ca oon o CA1 exci a o y hippocampal bou ons making con ac s wi h a
pos synap ic neu on. (B) Ske ch o ou ce ebella mossy ibe s con ac ing a g anule cell. (C) Illus a ion o a single mo o ne e e minal inne a ing a muscle ibe .
(D) Scaled ep esen a ion o he mean sizes o single Ac i e Zones (AZs; pu ple ci cles) and hei co esponding pe i-AZs (g ay ci cles) in h ee synapse ypes ( he
neu omuscula junc ion (NMJ), he ce ebella mossy ibe -g anule cell, and he hippocampus), a es (g ay ci cles), and a e usion o all synap ic esicle (SVs)
docked a each AZ (b oken line ci cles). No e he simila i y in he pe i-AZ a eas among di e en synapses. (E) Time cons an o luo escence eco e y ( au) s.
numbe o s imuli (1, 50 and 100 AP) in mouse mo o ne e e minals (whi e symbols adap ed om Taba es e al., 2007; Ga ield e al., 2009), and in small
hippocampal p esynap ic bou ons om neu ons in cul u e, black symbols adap ed om A mb us e e al. (2013).
a oom empe a u e, he ime cons an o endocy osis was
es ima ed o be 14–16 s a e 1 AP, and he same up o 100
ne e impulses (Figu e 1E;Sanka ana ayanan and Ryan, 2001;
Muelle e al., 2004; G anse h e al., 2006; Balaji and Ryan,
2007; Balaji e al., 2008). A he adul mouse NMJ, howe e ,
he endocy osis ime cons an , measu ed ex i o, was epo ed
o be h ee old as e (4–5 s a e 50 APs; Figu e 1E;Taba es
e al., 2007; Cano e al., 2012, 2013). When hese measu emen s
we e done a physiological empe a u e, he di e ence pe sis ed
be ween hese wo synapses. Fo example, a e a single AP he
ime cons an o endocy osis is <2 s a he NMJ (Figu e 1E;
Ga ield e al., 2009), and be ween 6–15 s a hippocampal
small bou ons (Figu e 1E;Balaji e al., 2008; A mb us e e al.,
2013).
Besides he di e ence in he p e alen mode o endocy osis
in dis inc synapses ypes, di e ences in he modula ion o
memb ane ecycling a e also p obable. Calcium is a majo
modula o o endocy osis in small and la ge synapses. Howe e ,
depending on he spa io empo al p o ile o he calcium
inc emen a elease si es, which in u n, depends on he densi y
o calcium channels, he ac i i y o kinases and phospha ases,
and he amoun and dis ibu ion o he di e en calcium bu e s,
he ou come may a y. E en mo e, he modula ion p ocess is a
e y dynamic p ocess in he same synapse. In small hippocampal
bou ons, he endocy osis kine ics is accele a ed when s imulus
s eng h inc eases om 1 o 25 APs and hen p og essi ely slows
o s imulus >25–100 APs (A mb us e e al., 2013). One o
he mechanisms by which calcium could accele a e endocy osis
is he calcineu in-dependen dephospho yla ion o he p o eins
implica ed in endocy osis, known as dephosphins, which include
dynamin, synap ojanin, amphiphysin, AP-2, AP-180, among
o he s (Ma ks and McMahon, 1998; Cousin and Robinson,
2000). Fo example, he amoun o dynamin dephospho yla ed
de e mines, in u n, he in e ac ion o dynamin wi h o he
p o eins o he endocy ic machine (Koch e al., 2011; A mb us e
e al., 2013; He man and Rosenmund, 2013; Wu e al.,
2014). The e o e, he endocy osis speed in di e en e minals
could be egula ed no only by he amoun o calcium in lux
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Cano and Taba es Small and La ge Synapse P ope ies
FIGURE 2 | The successi e s a es o he Release Si es (AZ) and he Pe i-AZ du ing synap ic ac i i y. Upon calcium en y du ing he ac ion po en ial (AP),
esicles use a he AZ (pu ple a ea) and elease neu o ansmi e in o he synap ic cle by exocy osis. The SV memb ane componen s a e eco e ed by endocy osis
h ough di e en pa hways: kiss-and- un, cla h in-dependen , bulk, and ul a as modes. The clea ing o he spen esicula ma e ial om AZ and pe iAZ a eas is
c i ical o he subsequen eloading o new esicles. Clea ance can ake place a he AZ, a he pe iAZ (g ay a ea), o ou side he pe iAZ. Fo simplici y, he shapes o
he AZ and pe iAZ a e shown as ec angles and a e no d awn o scaled. The wid hs o he a ows a e p opo ional o he speeds o he p ocess.
du ing each AP bu also by he exp ession le el, and spa ial
dis ibu ion, o he endocy ic molecula componen s in each
synapse ype.
‘‘Ho spo s’’ o endocy ic p o eins nea si es o exocy osis ha e
been desc ibed in la ge synapses, o example, in D osophila
(Es es e al., 1996; González-Gai án and Jäckle, 1997; Roos and
Kelly, 1998), snake (Teng e al., 1999) and mouse (Ga ield e al.,
2009) NMJs. In cen al synapses, an en ichmen o endocy ic
p o eins a he edges o he AZs is also p obable, as sugges ed
by he obse a ion o ul a as endocy osis in his loca ion a
hippocampal bou ons (Wa anabe e al., 2013b).
The mechanism by which calcium slows endocy osis a e
p olonged s imula ion emains unclea . I could be ha calcium
inc eases he a e o endocy osis du ing s imula ion un il
he capaci y o he endocy ic machine y becomes insu icien
(Sanka ana ayanan and Ryan, 2000). A es , he endocy ic
machine y is abundan (Roos and Kelly, 1999), bu a e se e al
ounds o ac i i y, he consump ion o he endocy ic p o eins
may slow he p ocess.
The exis ence o a ‘‘cla h in-coa ed eady- o-go pool
o esicles’’ a es has been sugges ed a he og NMJ
(Mille and Heuse , 1984). The o igin o hese s anded
p o ein spo s is, howe e , no clea . Do hey come om
used esicles ha ne e los hei iden i y o , al e na i ely,
esul a e p o ein in e mixing and so ing? A deg ee
o in e mixing be ween esh and old esicle p o eins
has been p oposed o occu in hippocampal synapses
du ing phasic s imula ion gi en ha s anded, and newly
inco po a ed esicle p o eins a e bo h in e nalized du ing
compensa o y endocy osis (Fe nandez-Al onso e al., 2006;
Wienisch and Klingau , 2006); he longe he s imulus
du a ion, he g ea e he in e mixing. Howe e , i is also
possible ha endocy osis o bo h new and s anded p o ein
pa ches occu in pa allel wi hou p e ious in e mixing
(Opazo and Rizzoli, 2010). The de elopmen o new
ools will p o ide a deepe unde s anding o he esicle
memb ane componen s dynamics du ing phasic and sus ained
s imula ion.
F on ie s in Synap ic Neu oscience | www. on ie sin.o g 4May 2016 | Volume 8 | A icle 12
Cano and Taba es Small and La ge Synapse P ope ies
RELEASE SITE REUSE
Du ing ongoing synap ic ansmission, elease si es a e
epea edly used. Howe e , has he elease si e a e ac o y
pe iod a e use? Knowing he numbe o elease si es a
p esynap ic e minal has and he amoun o quan a eleased
du ing a ain o s imula ion, i is possible o es ima e he
mean minimum numbe o imes a si e is used and he ime
in e al be ween uses. Fo example, a he mouse NMJ, a ain
o 100 APs a 100 Hz, p oduces he usion o abou 3200 SVs,
which ep esen s he size o he whole RRP o esicles (1700)
plus abou 1500 mo e (Ruiz e al., 2011). I e oked elease
occu s only wi hin he limi s o an AZ, each one hos ing wo
elease si es (Nagwaney e al., 2009), and all elease si es a e
used a leas once, he ‘‘mean euse index’’ is, in his example,
1.88, esul ing om di iding cumula i e elease by he o al
numbe o elease si es (3200/1700). In his case, 88% o si es
elease, clea ou o esicula componen s, dock, p ime, and
elease again in less han 1 s. In he calyx o Held, a simila
ime o e-usage has been es ima ed du ing he i s second
o s imula ion a 100 Hz (Nehe , 2010). A highe equencies
o s imula ion, he p ocess could be e en as e . Such a apid
clea ance o he excess o memb ane a he elease si e could
be achie ed ei he by endocy osis in si u (kiss-and- un) o by
mo ing he esicula componen s o he pe iAZ om whe e
hey a e la e ecycled (Figu e 2). Howe e , when he memb ane
load is oo la ge, o example, du ing sus ained high- equency
s imula ion, he sys em becomes less e icien . An excessi e
memb ane accumula ion a he pe iAZ may in e e e wi h
he la e al mo emen o he used memb ane om he AZ o
he pe iAZ, e en be o e deple ion o SVs occu s, con ibu ing
o sho - e m dep ession (Nehe , 2010; Hua e al., 2013).
Rema kably, ansloca ion, docking, and p iming o esicles
du ing he pla eau phase ha ollows sho - e m dep ession can
also occu e y as du ing sus ained s imula ion. Fo example,
in he ce ebella mossy ibe e minal, his p ocess has been
sugges ed o ake 12 ms (Sa iane and Sil e , 2006), simila
o he elease si e echa ging ime in some ibbon synapses
(G iesinge e al., 2005). These obse a ions sugges ha , wi hin
a gi en synapse, no all he elease si es has he same capabili y
o being eused a high a es o sus ained s imula ion. The basis
o his he e ogenei y is no clea . I could be only appa en i
some esicles use ou side he ‘‘well s uc u ed’’ elease si es and
his speed up he p ocess (Zenisek e al., 2000; Nehe , 2010).
Ne e heless, i could also be due o molecula di e ences in
he molecula componen s in ol ed in docking/p iming o
he esicles, o spa ial a ia ion in he p obabili y o ‘‘in si u’’
endocy osis, and e en o dispa i ies in he eloci y a which
dis inc pe iAZs ansloca e esicula componen s o neighbo
egions be o e endocy osis.
SUMMARY
The s uc u al and unc ional p ope ies o p esynap ic e minals
a e p incipal de e minan s o he success ul ansmission o
in o ma ion in he ne ous sys em. Ne e e minals di e no
only in size bu also in he numbe , shape and dimensions
o hei AZs and pe iAZs, as well as in he magni ude o
he ecycling pool o SVs. La ge e minals ha e hund eds o
AZs and elease a la ge numbe o quan a in esponse o
s imula ion. Small e minals possess one o ew AZs and elease
a low numbe o quan a. The g ea e exocy ic esponse in la ge
e minals does no imply a highe load o esicula componen s
a he pe iAZs, qui e he con a y, he dis ibu ion o his
ma e ial in a la ge numbe o uni s p obably acili a es he
managemen o he memb ane excess un il endocy osis akes
place. The p e e ed mode o endocy osis used unde each
egime o ac i i y in each synapse ype is s ill con o e sial,
bu i seems o be as a e b ie s imula ion ains, a leas a
physiological empe a u e. Wi h sus ained epe i i e ac i a ion,
howe e , endocy osis becomes slowe , p obably due, among
o he ac o s, o he sa u a ion o he endocy osis mechanisms
and he subsequen accumula ion o esicula memb ane a he
pe iAZ. Rema kably, many p esynap ic e minals can sus ain a
small and almos cons an amoun o ac i i y (pla eau) upon
p olonged high- equency s imula ion, appa en ly suppo ed by
a subpopula ion o elease si es ha can ope a e as e han
o he s. I he basis o such he e ogenei y is a he le el o he AZ
p o eins o he pe iAZs o ganiza ion emains o be de e mined.
AUTHOR CONTRIBUTIONS
RC and LT concei ed and w o e he manusc ip .
ACKNOWLEDGMENTS
We a e g a e ul o Bill Be z o discussions and commen s on he
manusc ip . This wo k was suppo ed by g an s om he Spanish
Minis y o Science and Inno a ion (BFU2013–43763-P) and he
Ta iana Pe ez de Guzman Founda ion.
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