Understanding how neuronal circuits can be modified in the mature brain requires knowledge of the mechanisms by which synapses can be shifted from a relatively stable state to a dynamic condition that allows new synaptic growth or elimination. Here we have shown that rapid activity-dependent changes in synapse development and function can be induced at the Drosophila
glutamatergic NMJs in a process that depends on spaced stimulation, akin to changes observed in dendritic spines of hippocampal neurons in culture (Yao et al., 2006
). During the postembryonic period after initial formation of synaptic contacts, synaptic boutons continuously proliferate in conjunction with changes in postsynaptic target size (Griffith and Budnik, 2006
). Chronic increases in activity, induced by mutations in ion channels, significantly enhance the formation of new synaptic boutons (Budnik et al., 1990
; Schuster et al., 1996a
). Here we demonstrate that activity-dependent changes are not simply the result of a developmental elevation in overall neuronal excitability, but rather that the ability of synapses to respond to changes in activity through structural and functional dynamics is an acute process, which is rapidly established upon patterned stimulation. Further, we have identified an important downstream mechanism, the Wnt pathway, which links activity-dependent changes to structural and functional synaptic modifications.
Together, the findings that (1) activity can induce Wg release from presynaptic boutons, (2) patterned activity elicits acute changes in synapse function and structural dynamics in a Wg-dependent fashion, (3) increasing Wg secretion can bypass some of the activity requirements, (4) intensifying or blocking activity has a corresponding influence on DFz2C entry into the nucleus in the postsynaptic muscle cell, and (5) activity modifications can be completely or partially suppressed by modulating GSK-3β in the presynaptic cell, demonstrate that bidirectional Wg signaling is a key downstream mediator of activity-dependent synaptic plasticity.
We propose the following model on bidirectional regulation of synapse structure and function by the Wg pathway (). Spaced stimulation results in the release of Wg by presynaptic boutons, which binds to DFz2 receptors present both pre- and postsynaptically (Packard et al., 2002
). In the presynaptic compartment, Wg release likely regulates cytoskeletal dynamics through inhibition of Sgg activity, leading to synaptopod dynamics and formation of ghost boutons in a process that depends on transcriptional and/or translational activation. In the postsynaptic compartment Wg release activates the FNI pathway, resulting in DFz2C cleavage and import into the nucleus where it may induce the transcription of synaptic genes (). Disrupting the Wg pathway during synapse development dampens the maturation of new synaptic boutons resulting in NMJs containing fewer mature boutons as well as a larger number of undifferentiated ghost boutons (Ataman et al., 2006
; Packard et al., 2002
). While our results are consistent with this model, alternative possibilities must be also considered. For example, changes in Wg signaling might alter excitability (beyond the parameters examined in this study) and these changes might trigger parallel transduction pathways that modulate synaptic structure and function. In addition, it is unlikely that Wg alone is responsible for all activity-dependent mechanisms. For example, the participation of a retrograde BMP signaling pathway in the regulation of synaptic bouton proliferation is well-established (Marques, 2005
). It is highly likely that multiple signaling pathways, including Wg, BMPs and others collaborate in the orchestration of activity-dependent synapse modifications.
Acute changes in synapse structure and function
The most striking structural changes we observed were the de novo
formation of synaptopods and ghost boutons. Although the nature of synaptopods is unclear, they might represent an initial stage during synaptic bouton formation like the filopodia observed at dendritic spines in normal animals and in response to activity (Niell et al., 2004
; Yuste and Bonhoeffer, 2004
). However, in our studies we never observed a transition from synaptopod to bouton. Another possibility is that they might correspond to exploratory structures that convey a signal to pre- and/or postsynaptic sites. This function has also been suggested for dendritic filopodia (Dunaevsky and Mason, 2003
; Yuste and Bonhoeffer, 2004
) as well as for growth cone filopodia prior to target innervation (Kalil and Dent, 2005
Ghost boutons, on the other hand, represent rapid de novo formation of undifferentiated boutons. Their formation is not the result of retraction of mature boutons. They are found to contain synaptic vesicles, but virtually lack pre- and postsynaptic specializations. Our live imaging studies showed that these boutons could acquire postsynaptic GluR and presynaptic Brp clusters over a relatively long period after their initial formation.
The above morphological changes required transcription and/or translation, akin to late LTP and long-term memory (Kandel, 2001
), but the step(s) at which they are required remains unclear. Potential scenarios include an activity-dependent increase in wg
mRNA or Wg protein synthesis as observed in the mammalian brain (Wayman et al., 2006
). They could also involve the activation of alternative pathways such as PKA and CREB-dependent mechanisms (Davis et al., 1998
; Davis et al., 1996
; Wayman et al., 2006
Previous live imaging studies of wild type intact larval NMJs did not report the occurence of synaptopods and ghost boutons (Zito et al., 1999
). However, in that study a postsynaptic marker (mCD8-GFP-Sh) was used to label the NMJ, and thus synaptopods and ghost boutons would not have been observed.
We also observed a Wg-dependent potentiation of mEJP frequency after spaced stimulation. An increased mEJP frequency has also been observed at the embryonic NMJ upon high frequency stimulation, although the accompanying structural changes were not examined in that preparation (Yoshihara et al., 2005
). The potentiation of mEJP frequency that we observed did not result from the addition of ghost boutons, as ghost boutons only rarely contain active zones(Ataman et al., 2006
). In addition, it occurred without any change in the number of nc82/Brp puncta in existing boutons (data not shown), suggesting that this potentiation is unlikely to emerge from the recruitment of new active zones. However, nc82/Brp is thought to label just the T-bar component of the active zone (Kittel et al., 2006a
), and many active zones lack T-bars (Atwood et al., 1993
). Thus, this possibility cannot be completely ruled out. Alternatively, the increase in mini frequency might arise from unsilencing of existing synapses as shown in mammals (Yao et al., 2006
) or from changes in their intrinsic properties.
Potentiation of spontaneous release frequency has been widely implicated in synapse maturation (Zucker, 2005
). At the Xenopus
NMJ repetitive neuron stimulation also results in the potentiation of spontaneous synaptic activity which is associated with synapse maturation (Lo et al., 1991
). Expressing SynCaM, a homophilic cell adhesion molecule that drives synaptic maturation, also increases the frequency of spontaneous release (Biederer et al., 2002
). At the Drosophila
embryonic NMJ mutations that block the potentiation of spontaneous release frequency, such as mutants lacking both DGluRIIA and DGluRIIB, as well as mutations in syntaxin
, and synaptotagmin IV
, exhibit abnormal NMJ development. These mutants all show a lack of presynaptic maturation, as demonstrated by a maintained growth cone structure (Yoshihara et al., 2005
). In the mammalian nervous system spontaneous release has been shown to regulate postsynaptic local protein synthesis which is thought to stabilize synaptic function (Chung and Kavalali, 2006
). Thus, the mEJP frequency potentiation observed here may play an initial role in postsynaptic maturation.
Importantly, spaced stimulation also induced a small but significant increase in mEJP amplitude that did not depend on Wg. Thus, activity is likely to regulate additional Wg-independent pathways. For example, elevated Ca++
induces the mobilization of vesicles to release sites, thereby increasing the number of active zones containing more than one docked vesicle, and thus eliciting multi-quantal release (Koenig et al., 1993). Recent studies have also suggested an activity-dependent increase in the size of synaptic vesicles (Steinert et al., 2006
). However, the change in mEJP amplitude was not accompanied by modifications in the amplitude of evoked responses, as expected if quantal size was larger, and further, we did not see a change in quantal content as determined by failure analysis.
Activity-dependent Wg release and role of Wg in functional and structural synaptic changes
Wg secretion was also found to be regulated by activity. Spaced depolarization increased the levels of Wg at the postsynaptic area, and conversely, temporally blocking activity in parats1 and by expressing ShiDNts in motorneurons decreased secreted Wg. Given that diminishing wg gene dosage prevented the rapid activity-dependent changes, these results suggest that Wg operates downstream of activity to promote these changes. This conclusion is further supported by the observation that increasing Wg secretion by overexpressing Wg in motorneurons, or activating the presynaptic Wg pathway by expressing a SggDN in motorneurons, partially bypassed the requirement for activity.
Notably, we found that activity-dependent Wg release did not decrease presynaptic Wg levels. In contrast, reducing Wg release through parats1
diminished Wg levels in presynaptic boutons. This is in agreement with recent studies documenting activity-dependent trafficking of peptidergic vesicles at the Drosophila
NMJ (Shakiryanova et al., 2005
; Shakiryanova et al., 2006
). In resting terminals peptidergic vesicles are relatively immobile, but activity induces a rapid mobilization of these vesicles to active terminals. Alternatively (or in addition), activity (or lack thereof) might influence the transcription and/or translation of Wg, consistent with the requirement of transcription and/or translation in ghost bouton formation.
Wnts and activity at mammalian synapses
Tetanic stimulation in hippocampal slices induces NMDA receptor-dependent release of Wnt3a by postsynaptic cells, the translocation of β-Catenin into the nucleus, and the upregulation of Wnt target genes (Chen et al., 2006
). Further, altering Wnt signaling levels caused corresponding changes in the magnitude of LTP (Ahmad-Annuar et al., 2006
; Chen et al., 2006
). Members of the Wnt pathway are also involved in activity-dependent dendritic arborization (Wayman et al., 2006
; Yu and Malenka, 2003
). Activation of an NMDA receptor-and Ca++
-dependent pathway resulted in CREB responsive transcription of Wnt-2, through activation of CaM kinase kinase (CaMKK), that coupled neuronal activity with dendritic development (Wayman et al., 2006
). Similarly, other studies suggest that the enhancement of dendritic growth induced by depolarization requires β-Catenin and an increased Wnt release (Yu and Malenka, 2003
To date, mammalian Wnts have been shown to be secreted by postsynaptic cells (Ciani and Salinas, 2005
). However, multiple members of the Wnt family exist both in Drosophila
and in mammalian systems, and different members might be released by different synaptic compartments. Alternatively, the anterograde, retrograde, or autocrine nature of Wnt signaling at synapses might have changed through evolution.
Notably, spaced, but not massed stimulation of cultured hippocampal neurons and dentate gyrus explants resulted in the persistent extension of postsynaptic filopodia and spine-like structures in dendrites (Wu et al., 2001
). As in our experiments, the induction of these structures depended on calcium and did not begin to appear until the 3rd
spaced stimulation cycle (Wu et al., 2001
). Spaced depolarization also led to an increase in mEPSC frequency thought to emerge from the activation of silent synapses (Yao et al., 2006
). Our findings in this study indicate that the cellular processes underlying rapid activity-dependent changes in synaptic structure and function appear to be conserved in presynaptic arbors of the NMJ.
Our results also implicate presynaptic GSK-3β in the presynaptic compartment during rapid activity-dependent changes at the larval NMJ. GSK-3β is known to regulate microtubule and actin cytoskeletons. In the case of microtubules, it phosphorylates MAP1B and tau, thereby influencing microtubule stability (Goold and Gordon-Weeks, 2004
). In agreement with those observations, GSK-3β/Sgg phosphorylates the Drosophila
MAP1B-related protein Futsch (Gogel et al., 2006
), and Futsch is required for Sgg function in synaptic growth (Franco et al., 2004
). Further, in sgg
mutants the number of bundled/stable microtubule loops within synaptic boutons were dramatically increased, exactly the opposite phenotype of wg
mutants (Franco et al., 2004
; Packard et al., 2002
In summary, our studies demonstrate that rapid modifications in synapse structure and function can be elicited in glutamatergic synapses at the larval NMJ, and identify Wg signaling as a critical effector of activity-dependent synaptic plasticity. These studies also provide a prominent in vivo model system to examine structural and physiological consequences of acute activity in a genetically tractable organism.