22494 ftp

Journal of Neuroscience Research 89:3–12 (2011)

Mini-Review
Silent Synapses in Neuromuscular Junction
Development
Josep Tomas,* Manel M. Santafe, Maria A. Lanuza, Neus Garcıa, Nuria Besalduch,
` ´ ´
`
and Marta Tomas
`
Unitat d’Histologia i Neurobiologia (UHN), Facultat de Medicina i Ciencies de la Salut,
Universitat Rovira i Virgili, Reus, Spain

In the last few years, evidence has been found to suggest SYNAPSE LOSS IN THE NEUROMUSCULAR
that some synaptic contacts become silent but can be JUNCTION
functionally recruited before they completely retract during The neuromuscular junction (NMJ) is a classic model
postnatal synapse elimination in muscle. The physiological from which much of our understanding of synaptic transmis-
mechanism of developmental synapse elimination may be sion has emerged (Katz, 1996), including the calcium-depend-
better understood by studying this synapse recruitment. ent quantal nature of transmitter release and the response of the
This Mini-Review collects previously published data and postsynaptic receptors. This is also true for the different forms
new results to propose a molecular mechanism for axonal of nervous system plasticity. In general, the basic NMJ
disconnection. The mechanism is based on protein kinase mechanisms operate throughout the nervous system. Synapse
C (PKC)-dependent inhibition of acetylcholine (ACh) elimination during initial synaptogenesis occurs in the NMJs
release. PKC activity may be stimulated by a methoctr- (Thompson, 1985), as it does throughout the nervous struc-
amine-sensitive M2-type muscarinic receptor and by cal- tures (Bourgeois and Rakic, 1993). The skeletal muscle cells in
cium inflow though P/Q- and L-type voltage-dependent newborn vertebrates are transiently polyinnervated at a single
calcium channels. In addition, tropomyosin-related tyro- synaptic site by several motor axons (Redfern, 1970; Brown
sine kinase B (trkB) receptor-mediated brain-derived neu- et al., 1976; Ribchester and Barry, 1994). Figure 1A shows a
rotrophic factor (BDNF) activity may oppose the PKC- pool of polyinnervated NMJ from a P6 rodent levator auris
mediated ACh release depression. Thus, a balance longus (LAL) muscle. Most evidence shows that, in the first
between trkB and muscarinic pathways may contribute to postnatal days, differential activity among the axons determines
the final functional suppression of some neuromuscular which endings are lost because relatively inactive synapses are
synapses during development. V 2010 Wiley-Liss, Inc.
C
permanently removed by the activity elicited by more active
inputs (Jansen and Fladby, 1990; Sanes and Lichtman, 1999;
Key words: postnatal synapse elimination; voltage-
however, see Callaway et al., 1987). Synapse elimination is ac-
dependent calcium channels; muscarinic acetylcholine
tivity dependent, because it slows down or speeds up when
receptors; protein kinases; neurotrophins
total neuromuscular activity decreases or increases, respectively.
In addition, the axon terminals that fire coordinately with the
postsynaptic cells can be enlarged and strengthened, whereas
DEVELOPMENTAL SYNAPSE ELIMINATION asynchronous synapses can be weakened (Bi and Poo, 2001;
Favero et al., 2010). Finally, in the adult, endplates are inner-
The development of the nervous system involves vated by a single axon (Benoit and Changeux, 1975; O’Brien
an initially exuberant production of neurons that estab- et al., 1978). Thus, in a nonrandom, active process, strong syn-
lish excessive synaptic contacts and the subsequent
reduction in both neurons and synapses. This develop-
mental process consists of an initial synapse overproduc- Contract grant sponsor: MEC; Contract grant number: SAF 2008-02836;
tion to promote broad connectivity and a subsequent ac- Contract grant sponsor: Catalan Government; Contract grant number:
2009SGR01248.
tivity-dependent reduction in synapse number. This
allows connectivity to be refined and specificity gained. `
*Correspondence to: Dr. J. Tomas, Unitat d’Histologia i Neurobiologia
Hebbian competition between the nerve terminals of `
(UHN), Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i
axons with different activities seems to be the fundamen- Virgili, Carrer St. Llorenc num 21, 43201-Reus, Spain.
¸
E-mail: jmtf@fmcs.urv.es
tal characteristic of this process of synapse elimination,
which leads to the loss of roughly half of the overpro- Received 22 April 2010; Revised 23 June 2010; Accepted 11 July 2010
duced elements (Fields and Nelson, 1992; Sanes and Published online 20 September 2010 in Wiley Online Library
Lichtman, 1999; Mennerick and Zorumski, 2000). (wileyonlinelibrary.com). DOI: 10.1002/jnr.22494

' 2010 Wiley-Liss, Inc.

4 `
Tomas et al.

aptic connections decrease the effectiveness of other inputs and Most studies have found a progressive diminution of the neu-
weak inputs can neither support themselves nor eliminate other rotransmitter release capacity of the nerve terminals that are dis-
inputs. A genetic method of selectively inhibiting neurotrans- connected. At the same time, the postsynaptic receptors are
mission from one of two inputs to a single target cell shows reorganized. However, the causal relation between axonal re-
that more powerful inputs are strongly favored competitors traction and postsynaptic receptor changes are not fully under-
during synapse elimination (Jia et al., 1999; Buffelli et al., stood (Colman et al., 1997; Lanuza et al., 2002).
2003). However, local differential effectiveness rather than (or
in addition to) differential activity should be the key determi- OCCURRENCE OF SILENT SYNAPSES
nant of eventual success, because an axon that fails at one syn- DURING DEVELOPMENTAL ELIMINATION
apse can be successful at another (Keller-Peck et al., 2001), IN MUSCLE
which suggests very local involvement of the postsynaptic mus- We know that, in polyinnervated synapses, quantal
cle cell and postsynaptic (and glial cell)-derived trophic factors. responses clearly decrease in both size and number

Figure 1.

Journal of Neuroscience Research

Silent Synapses During Development 5

before axonal withdrawal is completed (Dunia and functional inputs for a large number of P6–P7 NMJs
Herrera, 1993; Colman et al., 1997). Neurotransmitter from rat LAL muscle. Then, we calculated the mean
release from the axon that survives is characterized by a value, defined as the polyinnervation index of the
greater quantal content, whereas the efficiency of the muscle (PI). The nerve was stimulated with increasing
input(s) that is removed decreases progressively, because intensity from zero until an endplate potential (EPP)
a small quantal content is associated with reduced postsy- was observed. If the size and latency of the EPP
naptic receptor density (Colman et al., 1997; Culican remained constant as the stimulus was increased, we
et al., 1998). The competitive interactions may affect concluded that the endplate was monoinnervated. In
neurotransmitter release first, and axonal detachment endplates with polyneuronal innervation, increasing the
may occur after the neurotransmitter release has been stimulus amplitude recruited one or more axons,
totally abolished. Does the release machinery still operate which produced a stepwise increment in the EPP
for some time after the cessation of neurotransmission? (Redfern, 1970). This compound EPP is built by
With this question in mind, we test the hypothesis that recruiting two or more axons. In many polyinnervated
some nerve terminals become silent before they com- synapses, the endings are separated not by the thresh-
pletely retract (and shed membrane-bound remnants; old but by the latency. To study the PI, we consider
Bishop et al., 2004), and before the complete end of the only those synapses in which the different inputs are
repression period but that they retain certain capabilities clearly separated, on successive stimulations (10–20
for evoked release that can be recovered. Understanding EPPs at 0.5 Hz), by the excitability threshold, the la-
the role that important molecules play in ACh release at tency, or both. In this way, reproducibility is guaran-
the time of synapse elimination may help in understand- teed and the individual inputs can be accurately iden-
ing this important mechanism. tified (see Fig. 1B).
In P6–P7 muscles, the PI was 1.63 6 0.14 with
FUNCTIONAL RECRUITMENT OF SILENT 47.92% 6 2.08% of monoinnervated junctions (Lanuza
SYNAPSES et al., 2001; Santafe et al., 2001). Figure 1C shows the
Imposed changes in synaptic activity can acceler- effect on PI of blocking or activating several key mole-
ate or delay the developmental synapse elimination cules involved in ACh release. An increase in PI can be
process (Jansen and Fladby, 1990). In most cases, devi- observed in the following circumstances: 1) specific
ations from the normal physiological tempo can occur block of calcium entry through L- and P/Q-type (but
for several hours or even days (Nelson et al., 2005). not N-type) voltage-dependent calcium channels
Here, we investigate the almost immediate response (VDCC) or high magnesium-mediated nonspecific cal-
(1 hr) of some motor nerve terminals that recover ac- cium inflow reduction; 2) M2-type muscarinic acetyl-
etylcholine (ACh) release by acute exposure to modu- choline autoreceptor (mAChR) block (but not M1, M3,
lators of the molecular pathways involved in neuro- and M4 subtypes block); 3) brain-derived neurotrophic
transmission. We used intracellular recording of the factor (BDNF) incubation but not stimulation with neu-
evoked synaptic potentials to observe the number of rotrophin-3 (NT-3), neurotrophin-4 (NT-4), or glial-
3
Fig. 1. A shows examples of polyinnervated NMJ from a P6 rodent that blocking the M2 mAChR, the L-type or the P/Q-type VDCC, or
levator auris longus (LAL) muscle. The electrophysiological raw data in the PKC and stimulating the neuromuscular preparation with exoge-
B shows two doubly innervated endplates (left; vertical bars 5 2 mV, nous BDNF show similar (increasing) PI response patterns. Calcium
horizontal bars 5 10 msec) and a polyinnervated NMJ (right; vertical inflow agents: Mg21, 5 mM; the N-type VDCC blocker x-conotoxin
bar 5 4 mV, horizontal bar 5 10 msec) in a P6 LAL muscle. The GVI-A (x-CgTx GVI-A), 1 lM; the L-type VDCC blocker Nitrendi-
superimposed traces show synapses with inputs that, on successive pine, 1 lM; the P/Q-type VDCC blocker x-agatoxin IV-A (x-Aga
stimulations, are clearly separated by the excitability threshold (V, IV-A), 100 nM; Ca21, 5 mM. Muscarinic agents: The nonspecific
graded nerve stimulation) or the latency, so the individual inputs can muscarinic blocker atropine (AT), 2 lM; the M2 blocker methoctr-
be accurately identified. In C, the diagrams are a graphic representation amine (METHOC), 1 lM; the M1 muscarinic blockers pirenzepine
that collectively show the pattern of action of the different agents used (PIR), 10 lM; muscarinic toxin 7 (MT-7), 100 nM; the M4 blockers
on changing polyinnervation index (PI). These substances block or tropicamide (TPC), 1 lM; muscarinic toxin 3 (MT-3), 100 nM; the
activate different molecules related to ACh release and PI can be M3 blocker 4-DAMP, 1 lM; the muscarinic agonists oxotremorine M
observed to increase in some circumstances. Some data are from Santafe (OXO-M), 1 lM; and oxotremorine T (OXO-T), 1 lM. Serine
et al. (2009) and Garcia et al. (2010). However, most values of the kinase agents: The PKC blockers calphostin C (CaC), 1 lM, stauro-
polyinnervation index have not been previously published: 4-DAMP, sporine (STP), 200 nM, and chelerythrine (CEL), 1 lM; the PKC
oxotremorine M, oxotremorine T, staurosporine, PMA, H-89, Sp-8- stimulator phorbol ester (PMA), 10 nM; the PKA blocker H-89,
BrcAMPs, K252-A, TrkB-IgG, Ac anti p75, PEP-5, NT-4, NT-3, 5 lM; the PKA stimulator Sp-8-BrcAMPs, 10 lM. Neurotrophin
GDNF. For these new experiments, n 5 5 muscles for each experi- agents: The tyrosine kinase blocker K252-A, 200 nM; the BDNF
mental group, minimum 15 fibers per muscle. The thick black line neutralizing fusion protein TrkB-IgG (TrkB-IgG), 1 lg/ml; the neu-
means ratio 1 (experimental PI/control PI) or ‘‘no effect.’’ The experi- tralizing antibody against p75NTR (Ac anti-p75), 5 lg/ml; the p75NTR
mental effect of the different substances is shown by the thick blue line. pathway blocker PEP-5, 1 lM; brain-derived neurotrophic factor
Green circles mean that the experimental PI is significantly different (BDNF), 50 nM; neurotrophin 4 (NT-4), 50 ng/ml; neurotrophin
from the control PI (P < 0.05), and blue squares mean that there is no 3 (NT-3), 200 ng/ml; glial-derived neurotrophic factor (GDNF),
difference (P > 0.05). SEMs are eliminated for clarity. It can be noted 200 ng/ml. Scale bar 5 10 lm.


6 `
Tomas et al.

derived neurotrophic factor (GDNF); and 4) protein
kinase C (PKC) block but not protein kinase A (PKA)
inhibition. Interestingly, PI cannot be reduced below
the control value (which may indicate that the with-
drawal of supernumerary axon terminals is accelerated)
by incubation with several substances that are known to
produce effects on neurotransmission opposite to those
that increase PI: 1) increased calcium influx with high
external calcium (5 mM), 2) activation of all mAChRs
with oxotremorine M or oxotremorine T, 3) increased
PKC activity with PMA, and 4) inhibition of endoge-
nous BDNF action (incubation with k-252a, trkB IgG,
an anti-p75 antibody, or pep-5). Thus, the mechanism
that represses ACh release involving calcium inflow,
mAChR, and PKC seems to operate at maximal
efficiency (but see below), whereas the neurotrophin
mechanism (presumibly activated by endogenous BDNF
in vivo) seems not to be able to counteract this mecha-
nism, because only stimulation with exogenously added
BDNF recruits silent inputs and increases PI.
FEATURES OF THE RECRUITED SILENT
SYNAPSES
What are the newly recruited EPPs like? We
attempted to observe EPP recruitment directly by show- Fig. 2. A shows the timing of the recruitment in monoinnervated
ing the time course of the effect of some agents on EPPs (open circles) and dually innervated (solid squares) junctions after
in the same (permanently impaled) fiber. In these single- VDCC block with nitrendipine or x-Aga IV-A (from Santafe et al.,
fiber experiments, singly or dually innervated endplates 2002, with permission). The histogram in B shows that the newly
were continuously monitored before and after the toxins recruited endings in the presence of CaC almost always had longer
or drugs were added to the bathing solution, and EPPs latencies than the nerve endings in mono- and dually innervated NMJs
were recorded every several minutes for a minimum of (some data from Santafe et al., 2007). C shows examples of representa-
tive single-fiber experiments. The superimposed records in i) show
60 min. previously undetectable EPPs that become manifest by incubation with
BDNF (arrowhead) only after stimulation in the same position, and in
Timing of the Recruitment of Silent Synapses ii) they show a randomly appearing MEPP in another endplate. Vertical
Figure 2A shows the timing of the recruitment bars 5 top, 2.5 mV; bottom, 3 mV. Horizontal bars 5 top, 8 msec;
in monoinnervated and dually innervated junctions af- bottom, 10 msec. In D, after the VDCC block, a newly recruited end-
ter VDCC block. With the single-fiber experiments, ing that interiorizes RH414 when stimulated in the presence of the dye
we found that the recruited EPPs can appear several (orange spot) appeared close to where all the active nerve terminals in
the plaque were initially detected (stained green with FM1-43 (from
minutes after CaC, a VDCC blocker, high-magnesium Santafe et al., 2002, with permission). Scale bar 5 50 lm.
Ringer, atropine, or BDNF had been added to the
bath (the mean time of all experiments was about
20 min). endings. Thus, their appearance and eventual disappear-
Recruitment Thresholds ance will be clearly observed. Interestingly, in the pres-
ence of CaC, the latency of the newly recruited endings
About 36% of silent endings have a low recruit- was almost always longer than that of the other endings
ment threshold (1–2 V), 57% have a medium recruit- (Fig. 2B).
ment threshold (2–4 V), and only 7% have a high
threshold (4 V). Once a recruited ending has appeared,
it can be elicited regularly, although failures do occur, as Spontaneous Potentials
they do in other EPPs. The low MEPP frequency in the newborn end-
plates (less than 1 min–1) does not perturb the identifica-
Latency tion of single, double, or multiple innervation or
The single-fiber experiments allowed us to charac- recruited EPPs. The recruited EPPs may be confused
terize precisely such electrophysiological parameters of with MEPPs because they are both small. However,
the newly recruited EPPs as their mean size and mean the MEPPs can appear randomly and spontaneously
latency. We found that the latency of the newly (Fig. 2Cii), whereas recruited EPPs appear only after
recruited endings can be either shorter (see Fig. 2Ci stimulation in the same position (Fig. 2Ci). To discount
for an example) or longer than that of the preexisting a postsynaptic effect of the substances used in our condi-



tions, we always recorded spontaneous miniature end- This pattern implies a transition from two to three or
plate potentials. Because MEPP frequency in the new- more inputs, matched by the rate of transition from one
born is low, we increased the frequency with high-po- to two inputs. Figure 3 also shows that the percentages
tassium Ringer. We made sure that MEPP amplitudes of NMJs according to the number of inputs in P6–P7
and postsynaptic resting membrane potentials were muscles incubated with certain substances (for instance,
always unaffected by the different substances and toxins BDNF after 1 hr, Fig. 3F, first histogram) are not signif-
used. Therefore, the drugs and toxins that we used acted icantly different from the percentage of NMJs in normal,
presynaptically and did not change the number of untreated P2–P3 animals (P 0.05, the number sign
AChRs in our conditions. The maximum percentage of means significant difference with respect to P2–P3).
variation in MEPP amplitude was 10.9% 6 2.37% for Thus, in these cases, the axonal recruitment restores the
MT-3 (P 0.05). The mean change in the MEPP am- number of functional inputs in the NMJs to that found
plitude in all experiments was 1.76 6 0.18. several days earlier in development (from 6–7 days to 2–
3 days postnatally). Interestingly (Fig. 3, yellow histo-
Potential Size gram), after 3 hr in the presence of 50 nM BDNF, the
The mean ratio of the EPPs evoked by the two distribution of the number of inputs (which show con-
axons in well-defined, dually innervated endplates after siderable recruitment until 1 hr) completely returns to
blocking the L- or P/Q-type VDCC was 2. However, the control P6–P7 values. Thus, at least for BDNF,
the ratio was substantially greater between the preexist- recruitment is a brief, transitory effect that delays but
ing single EPP size in monoinnervated junctions and the does not impair the process of synapse elimination. Also,
recruited EPP (7), which indicated that the recruited Figure 3 (blue histograms) shows that, although no phar-
EPP was small. Moreover, the sizes of the recruited EPP macological treatment reduces PI significantly below the
from initially mono- and dually innervated synapses control value observed in P6–P7 animals (see Fig. 1C), a
were not different (on average, 0.87 mV 6 0.41 and significant partial acceleration of axonal elimination is
0.89 mV 6 0.35, respectively; P 0.05; Santafe et al., observed in high external calcium and more intensely in
2007). The mean MEPP size (mV) of newborn synapses the presence of the nonspecific muscarinic agonist oxo-
is 1.05 6 0.18. This value is not significantly different in tremorine T, because the three- and four-input junctions
comparison with the mean size of the recruited EPP (P are quickly reduced. This effect is just the opposite of
0.05), so the recruited axon can release the ACh con- that of axonal recruitment and indicates that the repres-
tent of a synaptic vesicle. sive mechanism that disconnects synapses may function
more intensely if stimulated.
Morphological Approach
We performed morphological experiments to visu- PROPOSED MECHANISM OF AXONAL
alize the recruited endings fluorescently (Santafe et al., DISCONNECTION
2001; Fig. 2D). We stained endplates with the activity- Figure 4 illustrates the pharmacological experiments
dependent and vital styryl dyes FM1-43 (green) and that were carried out to show the effect of a given sub-
RH414 (orange) and a-bungarotoxin fluorescently la- stance on PI during incubation with one of the other
beled with tetramethylrhodamine isothiocyanate. After 1 substances. The additive or occlusive effects between
hr of incubation with x-Aga-IVA, new orange spots these substances can be seen and inferences about the
(silent endings that interiorize RH414 when stimulated possible confluence of the action mechanisms can be
in the presence of the dye) appeared in the motor end- drawn. The diagram in Figure 5 illustrates the proposed
plate close to where all the active nerve terminals in the relation among mAChR, neurotrophins, PKC, and
plaque were initially detected (these spots are stained VDCCs in the process of developmental functional dis-
green with FM1-43). connection. We found that the individual effects of two
VDCC blockers, or a channel blocker and high magne-
DISTRIBUTION OF MOTOR ENDPLATES sium, on PI were not additive (Fig. 4A,B). This indicates
ACCORDING TO THE NUMBER OF INPUTS that calcium inflow per se is a main player in synapse
AFTER RECRUITMENT disconnection. However, the data show that CaC incu-
Figure 3 shows the distribution of NMJs according bation significantly increased PI above the level to which
to the number of functional axonal inputs after incuba- it had been increased by high magnesium (Fig. 4C) but
tion with representative substances used to investigate not above the level to which it had been increased by a
changes in PI. When a given substance induces the VDCC block (Fig. 4D). The difference between these
recruitment of silent terminals (and thus increases PI), two situations is that in high-magnesium media the
the percentage of singly innervated synapses after recruit- VDCCs are functional. This suggests that synaptic recov-
ment was significantly lower than in the P6–P7 control ery after the reduction of calcium entry in high magne-
(green histograms; the black histograms mean that the sium may be the result of calcium-dependent cPKC
corresponding substance does not change PI; see also inactivity, whereas the subsequent action of CaC may
Fig. 1C). Consistent with this observation, there was a lead to the direct complementary block of a calcium-in-
parallel increase in NMJs with more than one ending. dependent nPKC isoform. Thus, PKC activation (which


Fig. 3. Distribution of NMJs according to the number of functional histograms: when a given substance induces the recruitment of silent
axonal inputs after incubation with the representative substances used terminals (and thus increases PI). Black histograms: when the corre-
to investigate the changes in PI. In all cases, a comparison was made sponding substance does not change PI. Blue histograms shows that
with distributions at P2–P3 (#P 0.05 with respect to P2–P3) and P6– although the corresponding pharmacological treatments does not
P7 (*P 0.05 with respect to P6–P7). Some data are from Garcia et al. reduce PI (see Fig. 1C), a signiﬁcant partial acceleration of axonal elim-
(2010) and Santafe et al. (2009). However, most of the distributions of ination is observed because the three- and four-input junctions are
NMJs shown have not been previously published: x-CgTx GVI-A, quickly reduced. Yellow histogram: a particular situation when, after 3
methoctramine MT-7, MT-3, oxotremorine T, staurosporine, PMA, hr in the presence of 50 nM BDNF, the distribution of the number of
NT-4, NT-3 and PEP-5. For these new experiments, n 5 5 muscles inputs (which show considerable recruitment until 1 hr) completely
for each experimental group, minimum 15 ﬁbers per muscle. Green returns to the control P6–P7 values.


Fig. 4. Pharmacological experiments designed to show the effect of a ment, n 5 5 muscles, minimum 15 fibers per muscle. Values are
given substance on PI during incubation with one of the other sub- expressed as a percentage (mean 6 SEM) of EPP amplitude at time 0
stances. Thus, the additive or occlusive effects between these substan- (control). *P 0.05 with respect to control values (T 5 0 min). #P
ces can be studied. Most data are from Santafe et al. (2009). The data 0.05 with respect to T 5 60 min. Each line represents three to six
shown in F have not been previously published. For this new experi- single-fiber experiments obtained from different muscles.

was finally blocked by CaC) may be partially calcium in- Which extracellular signals from the local context
dependent. Nevertheless, the calcium channels them- of the activity-dependent axonal competition affect the
selves seem to be an important PKC target, because, PKC-VDCC mechanism in the synaptic contacts that
when they are blocked (see P/Q in Fig. 4D), CaC- are disconnected? Our experiments indicate that the
induced PKC inhibition no longer has any nerve ending BDNF-trkB (Fig. 5, point 5) and the ACh-mAChRs
recruiting activity. Thus, we hypothesize that PKC con- (Fig. 5, point 6) pathways may have a leading role.
tributes at least partially to synapse disconnection by
modulating VDCC operation (Fig. 5, point 1). Calcium Involvement of BDNF
entry also modulates PKC involvement in the pathway As with CaC, Figure 4G shows that BDNF can
(Fig. 5, point 2). In this regard, it is known that the extend the effect of high magnesium and also consider-
VDCCs are targets for PKCs that may regulate the effi- ably increase functional polyinnervation (mainly the tri-
cacy of calcium permeation. The influence of both PKC ple synapses). However, previous incubation with
and PKA on ACh release depends on the P/Q-type nitrendipine or CaC (Fig. 4H,I, respectively) completely
VDCC (Santafe et al., 2006), and PKC can regulate N- impairs any additional effect of BDNF. Thus, only when
type (Yokoyama et al., 2005) and L-type (Arenson and the first incubation is performed in high magnesium can
Evans, 2001) channels. Finally, calcium entry may have a both CaC and BDNF extend recruitment above that
direct influence on synapse disconnection, probably observed in high magnesium. This suggests that the
through a calcium-dependent protease system (Fig. 5, pathway turned on by BDNF may inhibit the PKC
point 3), and/or indirectly through a cPKC-mediated mechanism or at least nPKC, because CaC seems to do
phosphorylation in another intermediary step leading to in high magnesium media. These results indicate that
synaptic withdrawal (Fig. 5, point 4). incubation with exogenous BDNF (but not any other


10 `
Tomas et al.

sall, 1998; Nathanson, 2000). Both intracellular pathways
can modulate the VDCC.
We believe that the involvement of presynaptic
mAChRs in the elimination process may allow direct
interaction between nerve endings through differential
activity-dependent ACh release. Thus, the most active
ending may directly punish endings that are less active
(ACh-filled arrow in Figure 5 indicates an ACh stream
coming from a strong nerve terminal). There is some
evidence to suggest that, in synapse elimination, active
synapses prosper by punishing their inactive neighbors
(see, e.g., Thompson, 1985). Axons may compete by
generating activity-mediated signals to destabilize directly
synaptic sites associated with other inputs. The with-
Fig. 5. Proposed relation among M2-type mAChR, the BDNF-trkB
drawal process is spatially regulated so that the branches
pathway, PKC and L- and P/Q-type VDCCs in the control of the of an axon that are nearest (50 lm) to the competitor’s
ACh release mechanism, and neurotransmission from the axonal territory are removed before the more distant branches
inputs in the process of developmental functional disconnection. undergo retraction (Gan and Lichtman, 1998). This short
Taking all the findings into consideration, we believe that the best distance is compatible with a local diffusion of ACh
interpretation of the results is as illustrated here. The green arrows within the common synaptic gutter between competing
indicate a stimulating action, and the red arrows indicate an inhibi- endings. We have observed that the different axonal
tory one. See text for explanation. inputs to a given NMJ in the LAL muscle are generally
intermingled (indeed, they share the same postsynaptic
gutter of 10 lm) in the same endplate site, especially
neurotrophin or the neurotrophic cytokine GDNF, in at the beginning and in the first half of the elimination
gray in Fig. 5) has the same effect as PKC and L-type process. However, although one neuron propagates the
VDCC block. Thus, BDNF may modulate the PKC- same activity pattern and level to all its axonal branches,
VDCC intracellular cascade that, when fully active in the competitive success of these branches can vary
certain motor nerve terminals, leads to synapse discon- greatly in different polyinnervated junctions (Keller-Peck
nection. We observed that the blockade of the trkB re- et al., 2001). This suggests local interactions that involve
ceptor (but not of p75NTR signalling) prevents exoge- the postsynaptic muscle cell. A target-derived neurotro-
nous BDNF-induced recruitment (Garcia et al., 2010). phic factor may oppose the M2 mAChR and PKC-
It seems as if the silent endings have lost the p75NTR mediated depressing action on release. Thus, we
signalling but not the trkB pathway, which may remain hypothesize that there is a displacement of the balance
precariously coupled to release, because resources for between both BDNF-trkB and muscarinic pathways that
neurotransmission progressively decrease as synapse elim- might contribute to the final functional suppression of
ination progresses. However, as previously stated, only some neuromuscular synapses during development.
added BDNF recruits silent inputs; preventing the action
of endogenous BDNF in vivo (by blocking the trkB re-
ceptor or with the trkB-IgG chimera) does not change
PI (see Fig. 1C). These observations suggest that exoge- CONCLUSIONS
nous BDNF only shows the existence of an ineffective Our results suggest that there is a functional dis-
pathway, because the low activity-dependent production connection period before the complete morphological
of BDNF near poorly active nerve endings could not disconnection of synapses during development. We
counteract the PKC-VDCC inhibitory mechanism. identified a small number of important molecules among
those that may potentially modulate the functional syn-
aptic disconnection. We found that blocking the M2
Involvement of mAChR mAChR signaling cascade, the L-type or the P/Q-type
The specific block of P- or L-type VDCC or a VDCC, or the PKC or stimulating the neuromuscular
simple reduction in calcium inflow, PKC block, or stim- preparation with exogenous BDNF increases ACh
ulation with BDNF results in the same synapse recruit- release (just enough to be detected) in functionally silent
ment as M2-type mAChR block. However, the M2 axon terminals. These endings may become silent before
blocker methoctramine recovers no silent endings when they are completely (anatomically) retracted and before
it acts after VDCC block (the L-type in Fig. 4F), which synapse elimination. The calcium ions, VDCC,
indicates that the M2-mediated effect shares the pro- mAChR, and PKC may act through a common active
posed VDCC-PKC mechanism. Muscarinic receptors synapse elimination mechanism. BDNF separately may
are known to couple to G-proteins to stimulate phos- stimulate synapse generation, for instance, as it does in
pholipase C (and PKC) or inhibit adenylyl cyclase and the visual cortex (Cabelli et al., 1995). The low activity-
PKA (Caulfield, 1993; Felder, 1995; Caulfield and Bird- dependent production of endogenous BDNF near



poorly active nerve endings could not counteract the disassembly of the postsynaptic specialization and withdrawal of
mAChR-PKC-VDCC inhibitory mechanism. Schwann cell processes. J Neurosci 18:4953–4965.
We did not identify true silent synapses, structural Dunia R, Herrera AA. 1993. Synapse formation and elimination during
specializations for neurotransmission that do not produce growth of the pectoral muscle in Xenopus laevis. J Physiol 469:501–509.
Favero M, Buffelli M, Cangiano A, Busetto G. 2010. The timing of
a physiological response in the receiving cell although impulse activity shapes the process of synaptic competition at the neu-
they become fully functional upon the induction of ac- romuscular junction. Neuroscience 167:343–353.
tivity-dependent processes such as long-term potentia- Felder CC. 1995. Muscarinic acetylcholine receptors: signal transduction
tion (Atwood and Wojtowicz, 1999). We did, however, through multiple effectors. FASEB J 9:619–625.
observe functional synapses that became silent during a Fields RD, Nelson PG. 1992. Activity-dependent development of the
stereotyped developmental process that eliminates some vertebrate nervous system. Int Rev Neurobiol 34:133–214.
nerve endings before they completely retract. One prop- Gan WB, Lichtman JW. 1998. Synaptic segregation at the developing
erty of the silent synapses that we have described is the neuromuscular junction. Science 282:1508–1511.
rapid restoration (in minutes) of the release capacity after Garcia N, Santafe MM, Tomas M, Lanuza A, Besalduch B, Tomas J.
interfering with disconnection. Mammalian synapses 2010. Involvement of brain-derived neurotrophic factor (BDNF) in the
have been observed to appear and be turned over rapidly functional elimination of synaptic contacts at polyinnervated neuromus-
in both physiological and ultrastructural studies in tissue cular synapses during development. J Neurosci Res (in press).
Gasparini S, Saviane C, Voronin LL, Cherubini E. 2000. Silent synapses
culture (Haydon and Drapeau, 1995) and brain slices in the developing hippocampus: lack of functional AMPA receptors or
(Isaac et al., 1997; Rumpel et al., 1998; Choi et al., low probability of glutamate release? Proc Natl Acad Sci U S A
2000; Gasparini et al., 2000). 97:9741–9746.
Haydon PG, Drapeau P. 1995. From contact to connection: early events
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