BDNF signaling in correlation-dependent structural plasticity in the developing visual system

During development, patterned neural activity instructs topographic map refinement. Axons with similar patterns of neural activity, converge onto target neurons and stabilize their synapses with these postsynaptic partners, restricting exploratory branch elaboration (Hebbian structural plasticity). On the other hand, non-correlated firing in inputs leads to synapse weakening and increased exploratory growth of axons (Stentian structural plasticity). We used visual stimulation to control the correlation structure of neural activity in a few ipsilaterally projecting (ipsi) retinal ganglion cell (RGC) axons with respect to the majority contralateral eye inputs in the optic tectum of albino Xenopus laevis tadpoles. Multiphoton live imaging of ipsi axons, combined with specific targeted disruptions of brain-derived neurotrophic factor (BDNF) signaling, revealed that both presynaptic p75NTR and TrkB are required for Stentian axonal branch addition, whereas presumptive postsynaptic BDNF signaling is necessary for Hebbian axon stabilization. Additionally, we found that BDNF signaling mediates local suppression of branch elimination in response to correlated firing of inputs. Daily In vivo imaging of contralateral RGC axons demonstrated that p75NTR knockdown reduces axon branch elongation and arbor spanning field volume.


Introduction
Sensory experience during early development is crucial for the formation of precise topographic maps throughout the brain [1]. Visual stimulation drives action potential firing in retinal ganglion cells (RGCs) of fish and amphibians during the period when they first extend into the optic tectum, whereas in mammalian development, spontaneous waves of retinal activity drive RGC patterned activity [2][3][4][5]. Therefore, temporal correlation in firing patterns of RGCs is indicative of their mutual proximity in the retina, and the developing visual system uses this information to instruct structural and functional refinement of retinotopic and higher-order feature maps [6][7][8]. Concretely, co-activation of RGC inputs, read out by calcium flux through postsynaptic N-methyl-D-aspartate receptors (NMDARs), stabilizes retinotectal synapses and their axonal arbors [7,[9][10][11][12][13][14].
This correlation-dependent structural and functional stabilization is referred to as "Hebbian plasticity" [15]. In contrast, asynchronous firing of RGC axons with respect to their neighboring inputs leads to synaptic weakening, accompanied by an increase in axonal branch dynamics and exploratory growth, referred to as "Stentian plasticity" [7,16,17]. Previous in vivo imaging and electrophysiological recordings indicate that activity in neighboring axons promotes the Stentian synaptic weakening and branch elaboration of those axons that have not otherwise been stabilized by correlated firing and NMDAR activation [7,17].
Brain-derived neurotrophic factor (BDNF) has been implicated as a potent modulator of synaptic and structural plasticity throughout the brain [18]. BDNF can be synthesized and released in a constitutive or activity-dependent manner from axonal terminals and dendrites [19][20][21][22][23][24]. It can be released as an active precursor form, proBDNF, which exerts its function mainly via p75 NTR and sortilin signaling, or as the cleaved mature form (mBDNF), which signals mainly via TrkB and p75 NTR /TrkB [25][26][27][28]. Furthermore, conversion of proBDNF to mBDNF is critical for correlation-dependent synaptic strengthening [29,30]. The specifics of BDNF synthesis, secretion and receptor signaling create multi-layered regulation underlying the diversity of functions served by BDNF in the brain. For example, at the developing neuromuscular synapses, proBDNF signaling through presynaptic p75 NTR is required for axonal retraction, whereas mBDNF signaling through presynaptic TrkB leads to axonal stabilization [31,32]. In the developing retinotectal system, exogenous application of BDNF induces RGC axon branching and growth, whereas depletion of endogenous BDNF reduces presynaptic synaptobrevin punctum formation and axonal branch stabilization [33][34][35]. BDNF signaling through the TrkB receptor is necessary for Hebbian synaptic strengthening induced by visual conditioning in the Xenopus retinotectal system, whereas proBNDF signaling through p75 NTR facilitates synaptic weakening [12,36]. Overall, the literature to date suggests that BDNF could both be involved in increased exploratory growth (Stentian plasticity), as well as in structural and synaptic stabilization (Hebbian plasticity) in the optic tectum in

Xenopus.
In this study, we set out to test what aspects of BDNF signaling are respectively involved in Hebbian and Stentian structural plasticity. We took advantage of the existence of ectopic ipsilaterally projecting (ipsi) RGC axons in albino Xenopus laevis tadpoles, which typically consist of just one or two axons and which spontaneously occur in fewer than half of all albino and wildtype animals [7,37,38]. Since these ipsi RGC axons are not observed in all tadpoles, it is more likely that they are a result of rare errors in pathfinding, rather than constituting a functionally specialized class of RGCs.
Exploiting the presence of an ipsi axon, we used optical fibers to present light to each eye, visually stimulating the ipsi axon either in or out of synchrony with the contralateral eye from which the bulk of retinotectal input originates. Furthermore, we selectively knocked down either p75 NTR or TrkB in the ipsi axons, providing evidence by in vivo imaging for the presynaptic involvement of p75 NTR , and TrkB, in Stentian axonal branching and growth driven by uncorrelated activity. We further showed that Hebbian stabilization in response to correlated firing was blocked by application of an extracellular TrkB-Fc to prevent BDNF signaling. Daily imaging of contralaterally projecting RGC axons revealed that the p75 NTR receptor is required for axonal arbor growth and 3D expansion, in line with a role in Stentian plasticity. Our findings suggest that these opposing forms of plasticity both depend on neurotrophin signaling but have distinct sites of action and engage different receptors.

Stentian and Hebbian structural plasticity are mediated by distinct components of BDNF signaling
We performed in vivo 2-photon imaging of EGFP-expressing ipsi RGC axonal arbors in the optic tectum every 10 min for up to 5 h while subjecting the animals to a Darkness-Asynchronous-Synchronous (DAS) visual stimulation protocol based on Munz et al.
(2014) (Figs 1A and 1B). Ipsi RGC axons exhibit greater branch dynamics and growth when stimulated asynchronously with respect to the majority contra RGC inputs, but synchronous activation of the ipsi and contra axons results in arbor stabilization [7]. To examine the contributions of BDNF signaling, we employed three strategies: either intraventricular injection of TrkB-Fc to sequester released BDNF acutely, or coelectroporation of EGFP and antisense morpholino oligonucleotide (MO) against p75 NTR (p75-MO) or TrkB (TrkB-MO) in the eye to achieve presynaptic knock-down of these receptors in the RGCs (Fig 1A, S1 Fig). MOs were labeled with the red fluorescent dye lissamine which filled the RGC out to its axonal terminal, permitting knock-down to be confirmed by the presence of both EGFP and lissamine fluorescence in individual ipsi axons. We quantified changes in the rates of branch additions and losses per axon for each 10 min imaging interval throughout the DAS protocol (Figs 1B and 1C).
Importantly, in the optic tectum ipsilateral to the electroporated eye, only the ipsi RGC axon contains the MO, whereas the majority RGC inputs, originating from the contralateral eye, are unmanipulated.
Ipsi axons containing Ctrl-MO (Control) exhibited a Stentian increase in branch dynamics during asynchronous stimulation of the two eyes, compared to Hebbian stabilization induced by synchronous stimulation (Figs 1D-G), consistent with our previous report (S2 Fig) [7]. Our previous study showed the strongest increase in branch additions within the first hour of asynchronous stimulation, whereas the strongest increase in branch loss occurred during the second hour of asynchronous stimulation. Therefore, we focused on these two time periods. The higher rate of branch addition during asynchronous stimulation compared to synchronous stimulation was prevented in p75-MO axons (Figs 1D and 1F), suggesting a direct role of the low-affinity neurotrophin receptor in Stentian plasticity. TrkB-MO in the ipsi axon also prevented the increase in branch addition during asynchronous stimulation (Figs 1D and 1F).
However, the persistence of a relative difference in TrkB-MO axon branch additions between asynchronous and synchronous stimulation conditions suggests that although blocking axonal TrkB signaling may reduce activity-dependent branching, it does not fully eliminate the effects of correlated activity (S3 Fig A and C).
On the other hand, depletion of BDNF signaling by extracellular application of TrkB-Fc prevented the Hebbian stabilization of branch dynamics, which manifests as a suppression of new branch additions and losses during synchronous binocular stimulation (Figs 1D-G). A significant increase in branch additions during synchronous stimulation was observed exclusively in TrkB-Fc treated, but not in neurotrophin receptor knock-down RGC axons, implying that BDNF must act on targets other than the axon itself, presumably the postsynaptic cells where it has been shown to mediate synaptic plasticity [36], to induce Hebbian retrograde signals that inhibit formation of new axonal branches.

BDNF signaling helps suppress branch loss during synchronous stimulation
In the optic tectum, RGC axons continuously extend and retract processes in the neuropil during structural refinement of the developing retinotopic map. Axonal branch elimination occurs in parts of the arbor where synapses have failed to undergo stabilization, permitting axon pruning and topographic map refinement [8]. TrkB-Fc depletion of BDNF signaling led to an increase in the rates of axonal branch loss in response to synchronous stimulation compared to control ipsi axons, with even more branch elimination during synchronous than asynchronous stimulation (Figs 1E and 1G,   S3 Fig B and D). This effect on branch loss during synchronous stimulation was at least partially replicated by p75 knockdown in the ipsi axon. On the other hand, branch elimination rates were not elevated during synchronous compared to asynchronous stimulation in ipsi axons with TrkB-MO. These data show that BDNF release, likely involving the activation of presynaptic p75 NTR , contributes to the Hebbian suppression of branch loss that occurs in response to correlated firing.
Taken together these findings strongly implicate BDNF release in the Hebbian suppression of branch addition and elimination that occurs in response to synchronous firing of inputs. Moreover, presynaptic p75 NTR appears to be required for the differential response of the axon to asynchronous and synchronous activity, as the Stentian addition of new branches during asynchronous stimulation and the Hebbian suppression of branch elimination during synchronous firing were both lost in p75-MO cells, which responded similarly to the two stimulation conditions. On the other hand, presynaptic knockdown of TrkB did not eliminate differences in branch dynamic behaviors between synchronous versus asynchronous conditions.

BDNF signaling spatially restricts branch elimination events
Local action of BDNF has previously been shown to alter synaptic maturation and dendritic morphology in cells in close proximity to release sites [39][40][41][42]. We sought to further explore the role of BDNF signaling in the spatial organization of axonal branch addition and elimination events in response to patterned activity (Figs 2A and 2B). To assess whether remodeling events were spatially clustered, indicative of local signaling on the arbor, we extracted the pairwise distances for all addition and for all elimination events during darkness, asynchronous and synchronous stimulation (Fig 2C) and calculated a mean event pair distance for each stimulation period (Fig 2D, Fig S4C-D), shown for the example axon (Fig 2C). Differences in arbor size and shape may impact measurements of mean event pair-distance. We therefore performed Monte Carlo simulations of the locations of events on the arbor (Fig S4A-B) and used the simulated mean event pair-distances to correct for differences in arbor morphology for all analyses.
The corrected mean pairwise distance between elimination events was significantly lower during synchronous stimulation compared to darkness in control axons, due to fewer branch elimination events occurring far apart from each other during correlated firing induced by synchronous stimulation (Figs 2D and 2E). These data reveal that correlated activity causes branch eliminations to become restricted to a relatively smaller portion of the total arbor. Extracellular depletion of BDNF with TrkB-Fc and, to a much lesser extent, knockdown of the receptors in RGC axons, resulted in a redistribution of branch elimination events, no longer favoring the event proximity normally seen with synchronous stimulation (Fig 2E). In particular, there was a significant difference between pair distances of branch loss events during synchronous stimulation in TrkB-Fc and control animals. Interestingly, this spatial phenomenon was limited to branch elimination, as we observed no difference in mean pairwise distances of addition events in control axons across the stimulation periods (Fig 2F). These findings point to a model in which secreted neurotrophin can influence branch eliminations and stabilization within spatially constrained local zones of action. This suggests that the suppression of branch loss during synchronous activation, mediated by neurotrophin release (Fig 1G), acts within a spatially restricted part of the arbor, creating zones where branches are relatively protected from elimination (Fig 2B,D)

Axon arbor elaboration over days relies on p75 NTR
Overexpression of the truncated TrkB isoform, which blocks BDNF signaling by sequestering BDNF or forming non-functional TrkB dimers, has been shown to reduce RGC axonal arborization over a day [43]. We therefore set out to identify the roles of both p75 NTR and TrkB in long-term axonal arbor elaboration by performing knock-down of each of the receptors under conditions of normal visual experience. In this case, we performed daily imaging over 4 days of contralaterally projecting RGC axons coelectroporated with EGFP and p75-MO or TrkB-MO. (Figs 3A-C). RGC axons knockeddown for TrkB exhibited a significantly greater accumulation of branch tips compared to axons with p75-MO (Figs 3D and 3E). Furthermore, p75 NTR knock-down resulted in a significant decrease in the skeletal length of the axon compared to control axons (electroporated with Ctrl-MO) and RGCs electroporated with TrkB-MO (Fig 3F). In addition, binning terminal segments (Fig 3D) by length revealed that as axonal arbors became more complex over 4 days, the proportion of terminal segments categorized as short (1-5 µm) decreased in control and TrkB-MO axons, whereas it remained unchanged in the p75-MO axons, consistent with their failure to undergo progressive elaboration (Fig 3G). These findings indicate that presynaptic p75 NTR underlies new branch accumulation and the elongation of short filopodium-like branches over days, resulting in a more complex terminal arbor, whereas TrkB helps keep the arbor compact.

Roles of p75 NTR and TrkB in RGC axonal arbor span enlargement
The retinotopic map occupies a 3D volume within the tectum [14], and therefore a relevant metric of an axon's morphology and refinement is the volume of its arbor span.
We calculated arbor span volume for contralaterally projecting RGC axons by obtaining a measure of the convexity of the 3D reconstructed axonal arbor, which was used to define an enclosing volume whose border lies between a tight fit and a convex hull around the arbor, as described previously [44] (Figs 4A-C). We found that TrkB-MO arbors expand more rapidly over 4 days to occupy a greater volume in the optic tectum compared to p75-MO axons (Fig 4D-E). These observations suggest that p75 NTR in the RGC may mediate enlargement of the axonal arbor span volume, whereas TrkB may contribute to maintaining the arbor volume compact.

BDNF signaling in Hebbian suppression of axonal branch dynamics
Hebbian axonal branch stabilization induced by correlated firing is dependent on the activity of NMDARs [7,11]. Postsynaptic knock-down of NMDARs in optic tectal neurons results in increased rates of dynamic branch additions and retractions in RGC axons, suggesting the existence of a retrograde signal that promotes presynaptic axon arbor stabilization in response to NMDAR activation [13]. Activation of tectal NMDARs can induce the synaptic entry of calcium, leading to the activation of CaMKII in optic tectal neurons, which has been shown in live imaging experiments to mediate a retrograde modulation of presynaptic axonal dynamics and growth [45,46]. The identity of this putative retrograde signaling factor remains unclear, but several lines of evidence have suggested BDNF as a feasible candidate retrograde signal [47][48][49][50]. In our experiments, presynaptic p75 NTR knockdown and sequestration of BDNF with TrkB-Fc both prevented the usual decrease in branch addition and loss caused by synchronous stimulation, suggesting that BDNF could act through activation of presynaptic p75 NTR in Hebbian plasticity. However, the robust effects of sequestering extracellular BDNF were at best only partially replicated by axonal p75 NTR knockdown (Fig 1D,F), suggesting that released BDNF likely also acts postsynaptically to drive synaptic changes that facilitate the release of additional retrograde signals (Fig 5). In support of this, in other systems, BDNF has been shown to be released downstream of postsynaptic NMDAR activation and to act as an autocrine signal mediating synaptic strengthening at dendritic spines [24,51]. This notion is supported in the retinotectal system by previous reports showing that BDNF downstream of NMDAR activation promotes the formation and stabilization of postsynaptic elements [35,36,52]. While a strength of our experimental design is the ability to knock down receptor in just the RGC axons while sparing postsynaptic partners in the tectum, an important caveat is that dendritic morphologies of electroporated RGCs in the retina are likely to be affected by receptor knockdown, as has been shown previously [53]. However, in that earlier study it was reported that retinal BDNF manipulation did not affect RGC axonal morphology in the tectum.

Presynaptic p75 NTR and TrkB in Stentian exploratory growth
Knock-down of p75 NTR and TrkB receptors prevented the Stentian increase in new branch addition that occurs when an axon does not fire in synchrony with its neighbors (Figs 1D and 1F) [7,17]. Furthermore, we found a decrease in branch tip accumulation over 4 days of repeated imaging in p75-MO axons (Fig 3E). Previous work demonstrated that when inputs surrounding an axon actively fire while that axon is quiescent, Stentian "exploratory growth" of the inactive axon takes place, suggesting the possible release of an intercellular growth-promoting signal by neighboring cells in the optic tectum [17].
From our experiments, we can conclude that axonal p75 NTR and possibly TrkB mediate the response to Stentian signals that promote branch addition and extension. TrkB may mediate a general increase in axonal branch addition during firing, as the difference between asynchronous and synchronous stimulation appears to be maintained in TrkB-MO, whereas p75 NTR appears to underlie the differential responses to asynchronous and synchronous stimulation (S3 Fig C). Putative p75 NTR ligands such as proBDNF, or other neurotrophins could be released directly by the neighboring axons, indirectly by the postsynaptic neurons or even by local glia (Fig 5A-B). Further investigation is required to reveal the identities and the exact sites of release of these potential ligands. ProBDNFinduced activation of p75 NTR has been shown to promote synaptic weakening both in Xenopus tadpoles and in mice [27,29,36], consistent with our previous finding that exploratory axon growth is accompanied by a marked reduction in synaptic strength [7].
In addition, our results are in line with previous reports showing that injection of BDNF in the optic tectum drives a robust increase in RGC axonal branch number, an effect which could be the result of activation of either TrkB or p75 NTR [33]. Previously, Xenopus RGC axons electroporated with TrkB.T1 have been shown to exhibit an increase in axonal branch addition and a decrease in the numbers of mature synapses [43], an observation consistent with our TrkB-Fc results. In particular, because those studies were performed by confocal imaging using fluorescence excitation light that would be visible to the tadpole, the conditions during imaging in those experiments would most likely resemble our synchronous stimulation paradigm.
In our daily imaging experiments of contralaterally projecting axons, knock-down of TrkB and p75 NTR gave opposite effects on axon arbor growth and branch formation after several days (Figs 3,4). This observation stands in contrast to our data on rapid branch dynamics in ipsi axons where both p75-KO and TrkB-KO manipulations appear to have decreased branching in response to asynchronous visual stimulation. One must consider, however, that unlike ectopic ipsi axons, the contralateral RGC axons exhibit coarse topographic order within the optic tectum and thus, on average exhibit a pattern of firing that is relatively correlated with that of their neighbors [54]; [7,[9][10][11][12][13][14]). It is therefore likely that correlated neural activity dominates among contra axons, obscuring the effects of Stentian plasticity for all but the most imprecisely targeted stray axons over days. The short-term dynamic imaging of ipsi axons, which allowed us to systematically control the degree of correlation in RGC firing, unmasked roles for presynaptic p75 NTR and TrkB in contributing to Stentian structural plasticity through an increase in the rate of new branch addition (Figs 1D and 1F). overexpression [43] both profoundly alter rapid branch addition and loss rates without manifesting significant changes in arbor size at 24 h. Although it is tempting to infer that long-term arbor remodeling is the direct cumulative consequence of the rapid dynamic branch behaviors observed over minutes, it may be more appropriate to think of rapid dynamics as reflecting the activation of underlying plasticity mechanisms rather than the outcome of the refinement process itself. In the current study and in our previous work we observed that asynchronous stimulation led to enhanced ipsi axon branch dynamics within minutes, but that arbors that had previously been exposed to Hebbian stabilizing stimuli were more refractory to exploratory growth [7]. Thus, the dynamic mechanisms revealed by our unique experimental design, would be expected to produce a range of intermediate outcomes depending on the animal's recent and ongoing sensory experience, with more consistent changes emerging after several days of natural visual experience (Fig 3F, G, Fig 4).
This raises a potential caveat in our experimental design that should be considered. Although we believe the ipsi RGC axons to be the result of a serendipitous axon guidance error and thus not a unique class of RGC, an important difference between ipsi axons and the typical contralaterally projecting axons is their past history of correlation with neighboring inputs. We cannot exclude the possibility that ipsi axons, having developed for many days under atypical conditions of constant asynchronous visual experience, could have a very different molecular signaling or transcriptional profile compared to contralaterally projecting axons that experience primarily synchronous activity. It is at least reassuring that ipsi axons do not appear to be more susceptible than other RGCs to undergo apoptosis (p = 0.375, log-rank test for survival, n = 6 ipsi, 15 contra). In addition, although neurotrophin signaling has been strongly implicated in RGC survival and cell death in disease and development, we also found that p75-MO and TrkB-MO RGCs had survival profiles that were indistinguishable from control axons over 4 days of imaging (S5 Fig), indicating that at the stages when we performed our imaging experiments these RGCs did not experience excess apoptotic pressure.

Local action of BDNF signaling in Hebbian branch stabilization
Our analyses on the spatial distributions of addition and elimination of axonal branches revealed that correlated firing led to a decrease in the mean distances between elimination events compared to what occurs in darkness, specifically a loss of the longest pair distances, suggesting that the arbor is subjected to local influences on branch elimination (Figs 2D-F). In contrast, the mean distance between branch addition events stayed similar regardless of stimulation. These data are consistent with previous observations in tadpoles with binocularly innervated optic tecta showing that axonal branches are added uniformly across the arbor, but branch loss tends to occur in the territory where inputs from the opposite eye dominate [11]. A decrease in the mean pairwise distance of elimination events could occur through localized action of axonal branch elimination signals or through locally diffusible signals that protect axonal branches from being eliminated in parts of the arbor where the firing of the axon is better correlated with activity in its local postsynaptic partners. We found that disrupting BDNF signaling in the retinotectal system resulted in increased rates of activity-dependent branch elimination (Fig 1E and 1G) and these elimination events occurred more ubiquitously throughout the arbor (Fig 2E). Together, our data (Fig 1G and Fig 2E) suggest that release of BDNF at sites where inputs are mutually correlated confers local axonal branch stabilization, such that blocking BDNF signaling increases the relative frequency of elimination events, even under conditions of correlated neural activity.
These findings together with previous reports that BDNF increases the synaptic clustering on RGC arbors [35] suggest a model in which local BDNF signaling results in localized synaptic and structural stabilization (Fig 5).

Molecular mechanisms of axonal "competition" in the optic tectum
It has been shown that activity-dependent release of pro-neurotrophin acts on axonal p75 NTR to induce axonal pruning of outcompeted inputs in the peripheral nervous system, both in sympathetic neurons and at the neuromuscular junction (NMJ) [31,[55][56][57].
Conversely, "winners" of the competition are maintained and strengthened via TrkAdependent (sympathetic neurons) or TrkB-dependent (NMJ) mechanisms. In the central nervous system, where polyinnervation of postsynaptic partners is predominant, competition appears to occur differently. For example, the "loser" RGC axons do not undergo apoptosis or retraction in response to competition, but rather undergo synaptic weakening and exhibit more dynamic growth and branching ( [6][7][8]). Furthermore, our data for the retinotectal projection provide evidence that activity per se is not sufficient to stabilize the "winning" axon, but rather correlated activity inducing postsynaptic NMDAR activation appears to be required. Nevertheless, the involvement of p75 NTR on the "punished" axon appears to be a common mechanism. It has been suggested that the distinct morphological consequences on "punished" axons in the peripheral versus central nervous systems may be attributable to different sets of interacting receptors or downstream signaling molecules expressed in these different cells, which, for example, may lead to RhoA activation in the one case versus RhoA inhibition in the other [58]. Our study did not identify the specific ligands that act via TrkB and p75 NTR to produce the Stentian increase in branch dynamics, but the potency of extracellularly applied TrkB-Fc implies a released neurotrophin, most likely BDNF or its precursor proBDNF based on expression patterns in tectal development [36,59]. Future studies involving knock-down of p75 NTR and its interactors, including Sortilin and Nogo receptor, will be important to further elucidate the precise ligands and downstream signaling in Stentian versus Hebbian plasticity.
Our data suggest a model in which presynaptic signaling through p75 NTR and possibly TrkB receptors is required for Stentian exploratory branching, and in which postsynaptic BDNF signaling is necessary to drive Hebbian stabilization that results in suppression of new axon branch addition (Fig 5). Furthermore, released BDNF and presynaptic p75 NTR mediate Hebbian suppression of branch loss. Opposing pre-and postsynaptic effects on branch initiation could reflect differences in the levels of expression of TrkB and p75 NTR and their interactors in dendrites and axons [60][61][62][63].
Another possibility is that proBDNF may be released in response to neural activity either from the neighboring RGCs or from the postsynaptic tectal neuron to act on p75 NTR (Fig   5B), but can be further converted to mBDNF through NMDAR-dependent activation or release of proconvertases such as tissue plasminogen activator (tPA) or matrix metaloproteinase-9 (MMP-9), leading to preferential activation of TrkB during synchronous stimulation (Fig 5C) [64][65][66]. tPA mediates synaptic strengthening in the retinotectal projection in response to visual conditioning [36]. MMP-9 has been implicated as a molecular switch (via proBDNF-to-mBDNF cleavage) to convert axon pruning to stabilization in the Xenopus NMJ, and synaptic weakening to strengthening in rodents [30,31,67]. In the Xenopus optic tectum, MMP-9 is necessary for the visual experience driven increase in tectal neuron dendritic growth [68]. It is also plausible that the mode of BDNF release varies during asynchronous and synchronous stimulation, in line with reports proposing that tonic versus acute release of BDNF may differentially regulate the cell surface level of TrkB and thus distinctly affect neurite outgrowth [69][70][71].
In summary, our experiments have used a unique visual stimulation protocol and targeted knockdown of BDNF receptors to reveal that distinct receptor classes and sites of BDNF signaling underlie Stentian and Hebbian structural plasticity during development (Fig 5). Based on our data and the literature, we propose that presynaptic p75 NTR and possibly TrkB signaling promote Stentian exploratory growth of retinotectal axons in response to asynchronous activity (probably via proBDNF release).
Furthermore, correlated firing results in NMDAR-dependent release (or pro-conversion) of mBDNF that induces Hebbian synaptic strengthening in postsynaptic tectal neurons and delivery of retrograde stabilization signals that suppress new axonal branch addition and locally restrict axonal branch elimination (Fig 5A and 5C).

Accessibility
Any reagents generated for this study will be made available upon request to the lead contact. Alternatively, the requestor will be directed to a public repository tasked with distributing the reagent.

Xenopus laevis tadpoles
The experiments described in this study were approved by the Animal Care Committee

TrkB-MO validation experiments
Albino

p75-MO validation experiments
Due to a lack of Xenopus laevis p75 NTR -specific antibody, we took an indirect route to validate that the p75-MO knocks down Xenopus laevis p75 NTR . The p75 NTR sequence was cloned from cDNA from st. 24 Xenopus laevis tadpoles [72]. Agarose (Invitrogen, 16520). The imaging chamber contains two channels for fiber optics, one leading to each eye, a perfusion and spillover chambers, as previously described [17]. During the whole imaging session, the tadpoles were perfused with O2-bubbled 0.

Image Analysis
All multiphoton z-series were denoised using CANDLE software implemented in MATLAB (MathWorks) which relies on non-local mean filtering methods [73]. Denoised 3D stacks were then used to reconstruct axonal arbors using "Autopath" and "Autodepth" features in Imaris 6.4.2 (Bitplane) for daily imaging experiments and by manual tracing in Dynamo software [7], implemented in MATLAB (MathWorks), generously provided by Drs. Kaspar Podgorski and Kurt Haas (UBC). For morphometric analysis of daily imaged RGC axonal terminal branch points were extracted from Imaris 6.4.2. PyImarisSWC Xtension, implemented in Python, installed in Imaris 9.5.1, was used to export the Imaris reconstructions as swc-files. The exported node coordinates were converted from pixel to μm. The swc-files were imported using "trees toolbox" [74], which was used to classify axonal segments according to their order derived by the Strahler ordering method, using "strahler_tree" function [75][76][77]. Axonal segments are defined as axonal structure bordered by two branch points or by a branch and a terminal point. The length of axonal segments with Strahler number of one, referred to as terminal segments was extracted and the distribution of terminal segments binned by length was followed over 4 days. The length of all segments with Strahler number>1 was counted toward the total skeleton length. Axonal span volume was calculated after obtaining the convexity of the 3D reconstructed arbors with "convexity_tree" function and then using the value to calculate and feed a shrink factor into "boundary_tree" function of "tree toolbox" as described by [44]. From the dynamic imaging experiments ipsi RGC axon branch additions and eliminations between two consecutive time-series (10 min) were extracted and further normalizations were performed as described in each individual case in the figure legends.
In Fig 1F,  interval between two timepoints. Addition events were defined as the branch points of newly added branches between "t-10 min" and "t". Elimination events were defined as the terminal points of branches lost between "t" and "t+10 min". Rigid body transformation using manual landmarks in Dynamo was applied to align the timepoints of the reconstructed arbor. The coordinates of all the events throughout a stimulation perioddark, asynchronous or synchronous, were extracted and the pairwise distances between all the elimination and all the addition events within a stimulation period were calculated.
For one axon from the TrkB-Fc group, there was only one elimination event throughout the dark period, which prevented the calculation of mean elimination event pair distance for the dark period and further normalization. Thus, this cell was excluded from the elimination event analyses. To assess whether the changes in mean distances were explained by changes in axon arbor morphology, a randomization of the events on the axonal arbor was performed. To do that, the reconstructions in Dynamo were used to extract the coordinates of each node and a swc-file was exported for each timepoint of the imaging session. The reconstruction was then resampled using "resample_tree" from "trees_toolbox" such that the nodes composing the arbor were equidistant at 0.15 μm.
The number of observed addition events between "t-10 min" and "t" was randomly distributed on the axonal arbor at "t". The number of observed elimination events between t" and "t+10 min" was randomly assigned to the terminal points of the axonal arbor at "t".
Pairwise distances between the simulated addition or elimination events within a stimulation period was calculated and then the mean pairwise distance for stimulation period obtained. This randomization was repeated 100 times and the average simulated mean pairwise distance plotted on Fig S4A-B. The ratio of the observed (Fig S4C-D) and simulated (Fig S4A-B

Statistical analysis
Aligned rank transform for non-parametric factorial 2-way mixed design model with BDNF manipulation as between-subject and visual stimulation (Figs 1E-F and 2E-F, Figs S2, Fig S4) or time (Figs 3E-G and 4D) as within-subject factor was performed using ART tool package implemented in R [78]. After obtaining significant interaction, to assess statistical significance of simple main effects, either Kruskal-Wallis or Friedman's test, followed by pairwise post-hoc Dunn's tests corrected for multiple comparisons using the Benjamini, Krieger and Yekutieli (BKY) two-stage linear step-up procedure [79] were carried out using GraphPad Prism 9.0.0 and the results of the tests used for each experiment are described for each figure legend and in supplemental table 1. In some cases (Fig S3A-B, Fig 4E) only Kruskal-Wallis test, followed by pairwise post-hoc Dunn's tests corrected for multiple comparisons using the BKY two-stage linear step-up procedure were carried out. The axonal reconstructions and their spanning fields in Fig   4A-C were plotted using Plotly.      BDNF binds to postsynaptic TrkB which initiates a retrograde stabilization signal (of