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  1. 1. Journal of Neuroscience Research 89:3–12 (2011)Mini-ReviewSilent Synapses in Neuromuscular JunctionDevelopmentJosep 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, SpainIn the last few years, evidence has been found to suggest SYNAPSE LOSS IN THE NEUROMUSCULARthat some synaptic contacts become silent but can be JUNCTIONfunctionally recruited before they completely retract during The neuromuscular junction (NMJ) is a classic modelpostnatal 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 theThis Mini-Review collects previously published data and postsynaptic receptors. This is also true for the different formsnew results to propose a molecular mechanism for axonal of nervous system plasticity. In general, the basic NMJdisconnection. The mechanism is based on protein kinase mechanisms operate throughout the nervous system. SynapseC (PKC)-dependent inhibition of acetylcholine (ACh) elimination during initial synaptogenesis occurs in the NMJsrelease. 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 incium inflow though P/Q- and L-type voltage-dependent newborn vertebrates are transiently polyinnervated at a singlecalcium channels. In addition, tropomyosin-related tyro- synaptic site by several motor axons (Redfern, 1970; Brownsine kinase B (trkB) receptor-mediated brain-derived neu- et al., 1976; Ribchester and Barry, 1994). Figure 1A shows arotrophic factor (BDNF) activity may oppose the PKC- pool of polyinnervated NMJ from a P6 rodent levator aurismediated ACh release depression. Thus, a balance longus (LAL) muscle. Most evidence shows that, in the firstbetween trkB and muscarinic pathways may contribute to postnatal days, differential activity among the axons determinesthe final functional suppression of some neuromuscular which endings are lost because relatively inactive synapses aresynapses 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 whenreceptors; 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’Brienan initially exuberant production of neurons that estab- et al., 1978). Thus, in a nonrandom, active process, strong syn-lish excessive synaptic contacts and the subsequentreduction 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. Thisallows connectivity to be refined and specificity gained. ` *Correspondence to: Dr. J. Tomas, Unitat d’Histologia i NeurobiologiaHebbian competition between the nerve terminals of ` (UHN), Facultat de Medicina i Ciencies de la Salut, Universitat Rovira iaxons with different activities seems to be the fundamen- Virgili, Carrer St. Llorenc num 21, 43201-Reus, Spain. ¸ E-mail: jmtf@fmcs.urv.estal 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 2010duced elements (Fields and Nelson, 1992; Sanes and Published online 20 September 2010 in Wiley Online LibraryLichtman, 1999; Mennerick and Zorumski, 2000). (wileyonlinelibrary.com). DOI: 10.1002/jnr.22494 2010 Wiley-Liss, Inc.
  2. 2. 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 aremission 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 (orin addition to) differential activity should be the key determi- OCCURRENCE OF SILENT SYNAPSESnant of eventual success, because an axon that fails at one syn- DURING DEVELOPMENTAL ELIMINATIONapse can be successful at another (Keller-Peck et al., 2001), IN MUSCLEwhich suggests very local involvement of the postsynaptic mus- We know that, in polyinnervated synapses, quantalcle cell and postsynaptic (and glial cell)-derived trophic factors. responses clearly decrease in both size and number Figure 1. Journal of Neuroscience Research
  3. 3. Silent Synapses During Development 5before axonal withdrawal is completed (Dunia and functional inputs for a large number of P6–P7 NMJsHerrera, 1993; Colman et al., 1997). Neurotransmitter from rat LAL muscle. Then, we calculated the meanrelease from the axon that survives is characterized by a value, defined as the polyinnervation index of thegreater quantal content, whereas the efficiency of the muscle (PI). The nerve was stimulated with increasinginput(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 EPPnaptic receptor density (Colman et al., 1997; Culican remained constant as the stimulus was increased, weet al., 1998). The competitive interactions may affect concluded that the endplate was monoinnervated. Inneurotransmitter release first, and axonal detachment endplates with polyneuronal innervation, increasing themay 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 EPPfor some time after the cessation of neurotransmission? (Redfern, 1970). This compound EPP is built byWith this question in mind, we test the hypothesis that recruiting two or more axons. In many polyinnervatedsome 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 considerBishop et al., 2004), and before the complete end of the only those synapses in which the different inputs arerepression period but that they retain certain capabilities clearly separated, on successive stimulations (10–20for 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 beprocess (Jansen and Fladby, 1990). In most cases, devi- observed in the following circumstances: 1) specifications from the normal physiological tempo can occur block of calcium entry through L- and P/Q-type (butfor several hours or even days (Nelson et al., 2005). not N-type) voltage-dependent calcium channelsHere, 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 neurotrophictransmission. 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-3Fig. 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, orlevator 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. Calciumhorizontal bars 5 10 msec) and a polyinnervated NMJ (right; vertical inflow agents: Mg21, 5 mM; the N-type VDCC blocker x-conotoxinbar 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-Agastimulations, are clearly separated by the excitability threshold (V, IV-A), 100 nM; Ca21, 5 mM. Muscarinic agents: The nonspecificgraded 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 pirenzepinethat collectively show the pattern of action of the different agents used (PIR), 10 lM; muscarinic toxin 7 (MT-7), 100 nM; the M4 blockerson changing polyinnervation index (PI). These substances block or tropicamide (TPC), 1 lM; muscarinic toxin 3 (MT-3), 100 nM; theactivate different molecules related to ACh release and PI can be M3 blocker 4-DAMP, 1 lM; the muscarinic agonists oxotremorine Mobserved to increase in some circumstances. Some data are from Santafe (OXO-M), 1 lM; and oxotremorine T (OXO-T), 1 lM. Serineet 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 PKCoxotremorine 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. NeurotrophinGDNF. For these new experiments, n 5 5 muscles for each experi- agents: The tyrosine kinase blocker K252-A, 200 nM; the BDNFmental 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 p75NTRmental effect of the different substances is shown by the thick blue line. pathway blocker PEP-5, 1 lM; brain-derived neurotrophic factorGreen circles mean that the experimental PI is significantly different (BDNF), 50 nM; neurotrophin 4 (NT-4), 50 ng/ml; neurotrophinfrom 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.Journal of Neuroscience Research
  4. 4. 6 ` Tomas et al.derived neurotrophic factor (GDNF); and 4) proteinkinase C (PKC) block but not protein kinase A (PKA)inhibition. Interestingly, PI cannot be reduced belowthe control value (which may indicate that the with-drawal of supernumerary axon terminals is accelerated)by incubation with several substances that are known toproduce effects on neurotransmission opposite to thosethat increase PI: 1) increased calcium influx with highexternal calcium (5 mM), 2) activation of all mAChRswith oxotremorine M or oxotremorine T, 3) increasedPKC 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 mechanismthat represses ACh release involving calcium inflow,mAChR, and PKC seems to operate at maximalefficiency (but see below), whereas the neurotrophinmechanism (presumibly activated by endogenous BDNFin vivo) seems not to be able to counteract this mecha-nism, because only stimulation with exogenously addedBDNF recruits silent inputs and increases PI. FEATURES OF THE RECRUITED SILENT SYNAPSES What are the newly recruited EPPs like? Weattempted to observe EPP recruitment directly by show- Fig. 2. A shows the timing of the recruitment in monoinnervateding the time course of the effect of some agents on EPPs (open circles) and dually innervated (solid squares) junctions afterin 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 newlywere continuously monitored before and after the toxins recruited endings in the presence of CaC almost always had longeror drugs were added to the bathing solution, and EPPs latencies than the nerve endings in mono- and dually innervated NMJswere 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) show60 min. previously undetectable EPPs that become manifest by incubation with BDNF (arrowhead) only after stimulation in the same position, and inTiming 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 dyewe 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 (fromminutes 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 thebath (the mean time of all experiments was about20 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 endingsment threshold (1–2 V), 57% have a medium recruit- (Fig. 2B).ment threshold (2–4 V), and only 7% have a highthreshold (4 V). Once a recruited ending has appeared,it can be elicited regularly, although failures do occur, as Spontaneous Potentialsthey 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 confusedterize 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 spontaneouslylatency. We found that the latency of the newly (Fig. 2Cii), whereas recruited EPPs appear only afterrecruited endings can be either shorter (see Fig. 2Ci stimulation in the same position (Fig. 2Ci). To discountfor an example) or longer than that of the preexisting a postsynaptic effect of the substances used in our condi- Journal of Neuroscience Research
  5. 5. Silent Synapses During Development 7tions, we always recorded spontaneous miniature end- This pattern implies a transition from two to three orplate potentials. Because MEPP frequency in the new- more inputs, matched by the rate of transition from oneborn is low, we increased the frequency with high-po- to two inputs. Figure 3 also shows that the percentagestassium Ringer. We made sure that MEPP amplitudes of NMJs according to the number of inputs in P6–P7and 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 signAChRs 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 theMT-3 (P 0.05). The mean change in the MEPP am- number of functional inputs in the NMJs to that foundplitude 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 toblocking 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 buting 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 thefrom initially mono- and dually innervated synapses control value observed in P6–P7 animals (see Fig. 1C), awere not different (on average, 0.87 mV 6 0.41 and significant partial acceleration of axonal elimination is0.89 mV 6 0.35, respectively; P 0.05; Santafe et al., observed in high external calcium and more intensely in2007). 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 junctionscomparison 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 AXONALalize the recruited endings fluorescently (Santafe et al., DISCONNECTION2001; Fig. 2D). We stained endplates with the activity- Figure 4 illustrates the pharmacological experimentsdependent 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 otherbeled with tetramethylrhodamine isothiocyanate. After 1 substances. The additive or occlusive effects betweenhr 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 bein the presence of the dye) appeared in the motor end- drawn. The diagram in Figure 5 illustrates the proposedplate close to where all the active nerve terminals in the relation among mAChR, neurotrophins, PKC, andplaque 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 whichto the number of functional axonal inputs after incuba- it had been increased by high magnesium (Fig. 4C) buttion with representative substances used to investigate not above the level to which it had been increased by achanges in PI. When a given substance induces the VDCC block (Fig. 4D). The difference between theserecruitment of silent terminals (and thus increases PI), two situations is that in high-magnesium media thethe 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 cPKCcorresponding substance does not change PI; see also inactivity, whereas the subsequent action of CaC mayFig. 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 (whichJournal of Neuroscience Research
  6. 6. Fig. 3. Distribution of NMJs according to the number of functional histograms: when a given substance induces the recruitment of silentaxonal 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 thatwith distributions at P2–P3 (#P 0.05 with respect to P2–P3) and P6– although the corresponding pharmacological treatments does notP7 (*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 areNMJs shown have not been previously published: x-CgTx GVI-A, quickly reduced. Yellow histogram: a particular situation when, after 3methoctramine MT-7, MT-3, oxotremorine T, staurosporine, PMA, hr in the presence of 50 nM BDNF, the distribution of the number ofNT-4, NT-3 and PEP-5. For these new experiments, n 5 5 muscles inputs (which show considerable recruitment until 1 hr) completelyfor each experimental group, minimum 15 fibers per muscle. Green returns to the control P6–P7 values.
  7. 7. Silent Synapses During Development 9Fig. 4. Pharmacological experiments designed to show the effect of a ment, n 5 5 muscles, minimum 15 fibers per muscle. Values aregiven substance on PI during incubation with one of the other sub- expressed as a percentage (mean 6 SEM) of EPP amplitude at time 0stances. Thus, the additive or occlusive effects between these substan- (control). *P 0.05 with respect to control values (T 5 0 min). #Pces 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 sixshown 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 contextdependent. Nevertheless, the calcium channels them- of the activity-dependent axonal competition affect theselves seem to be an important PKC target, because, PKC-VDCC mechanism in the synaptic contacts thatwhen they are blocked (see P/Q in Fig. 4D), CaC- are disconnected? Our experiments indicate that theinduced PKC inhibition no longer has any nerve ending BDNF-trkB (Fig. 5, point 5) and the ACh-mAChRsrecruiting activity. Thus, we hypothesize that PKC con- (Fig. 5, point 6) pathways may have a leading role.tributes at least partially to synapse disconnection bymodulating VDCC operation (Fig. 5, point 1). Calcium Involvement of BDNFentry 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 withand PKA on ACh release depends on the P/Q-type nitrendipine or CaC (Fig. 4H,I, respectively) completelyVDCC (Santafe et al., 2006), and PKC can regulate N- impairs any additional effect of BDNF. Thus, only whentype (Yokoyama et al., 2005) and L-type (Arenson and the first incubation is performed in high magnesium canEvans, 2001) channels. Finally, calcium entry may have a both CaC and BDNF extend recruitment above thatdirect influence on synapse disconnection, probably observed in high magnesium. This suggests that thethrough a calcium-dependent protease system (Fig. 5, pathway turned on by BDNF may inhibit the PKCpoint 3), and/or indirectly through a cPKC-mediated mechanism or at least nPKC, because CaC seems to dophosphorylation in another intermediary step leading to in high magnesium media. These results indicate thatsynaptic withdrawal (Fig. 5, point 4). incubation with exogenous BDNF (but not any otherJournal of Neuroscience Research
  8. 8. 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 branchespathway, PKC and L- and P/Q-type VDCCs in the control of the of an axon that are nearest (50 lm) to the competitor’sACh release mechanism, and neurotransmission from the axonal territory are removed before the more distant branchesinputs in the process of developmental functional disconnection. undergo retraction (Gan and Lichtman, 1998). This shortTaking all the findings into consideration, we believe that the best distance is compatible with a local diffusion of AChinterpretation of the results is as illustrated here. The green arrows within the common synaptic gutter between competingindicate a stimulating action, and the red arrows indicate an inhibi- endings. We have observed that the different axonaltory 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, especiallyneurotrophin or the neurotrophic cytokine GDNF, in at the beginning and in the first half of the eliminationgray in Fig. 5) has the same effect as PKC and L-type process. However, although one neuron propagates theVDCC 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 varycertain motor nerve terminals, leads to synapse discon- greatly in different polyinnervated junctions (Keller-Pecknection. We observed that the blockade of the trkB re- et al., 2001). This suggests local interactions that involveceptor (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, wesignalling but not the trkB pathway, which may remain hypothesize that there is a displacement of the balanceprecariously coupled to release, because resources for between both BDNF-trkB and muscarinic pathways thatneurotransmission progressively decrease as synapse elim- might contribute to the final functional suppression ofination progresses. However, as previously stated, only some neuromuscular synapses during development.added BDNF recruits silent inputs; preventing the actionof endogenous BDNF in vivo (by blocking the trkB re-ceptor or with the trkB-IgG chimera) does not changePI (see Fig. 1C). These observations suggest that exoge- CONCLUSIONSnous 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 morphologicalof BDNF near poorly active nerve endings could not disconnection of synapses during development. Wecounteract 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 M2Involvement 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 neuromuscularsimple reduction in calcium inflow, PKC block, or stim- preparation with exogenous BDNF increases AChulation with BDNF results in the same synapse recruit- release (just enough to be detected) in functionally silentment as M2-type mAChR block. However, the M2 axon terminals. These endings may become silent beforeblocker methoctramine recovers no silent endings when they are completely (anatomically) retracted and beforeit 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 activeposed VDCC-PKC mechanism. Muscarinic receptors synapse elimination mechanism. BDNF separately mayare known to couple to G-proteins to stimulate phos- stimulate synapse generation, for instance, as it does inpholipase 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 Journal of Neuroscience Research
  9. 9. Silent Synapses During Development 11poorly active nerve endings could not counteract the disassembly of the postsynaptic specialization and withdrawal ofmAChR-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 duringspecializations 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 ofa 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 transductiontion (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 thestereotyped 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 developingerty 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 thehave 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). 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