Impact of maternal n-3 polyunsaturated fatty acid deficiency on dendritic arbor morphology and connectivity of developing Xenopus laevis central neurons in vivo.

Docosahexaenoic acid (DHA, 22:6n-3) is an essential component of the nervous system, and maternal n-3 polyunsaturated fatty acids (PUFAs) are an important source for brain development. Here, the impact of DHA on developing central neurons was examined using an accessible in vivo model. Xenopus laevis embryos from adult female frogs fed n-3 PUFA-adequate or deficient diets were analyzed every 10 weeks for up to 60 weeks, when frogs were then switched to a fish oil-supplemented diet. Lipid analysis showed that DHA was significantly reduced both in oocytes and tadpoles 40 weeks after deprivation, and brain DHA was reduced by 57% at 60 weeks. In vivo imaging of single optic tectal neurons coexpressing tdTomato and PSD-95-GFP revealed that neurons were morphologically simpler in tadpoles from frogs fed the deficient diet compared with the adequate diet. Tectal neurons had significantly fewer dendrite branches and shorter dendritic arbor over a 48 h imaging period. Postsynaptic cluster number and density were lower in neurons deprived of n-3 PUFA. Moreover, changes in neuronal morphology correlated with a 40% decrease in the levels of BDNF mRNA and mature protein in the brain, but not in TrkB. Importantly, switching to a fish oil-supplemented diet induced a recovery in DHA content in the frog embryos within 20 weeks and diminished the deprivation effects observed on tectal neurons of Stage 45 tadpoles. Consequently, our results indicate that DHA impacts dendrite maturation and synaptic connectivity in the developing brain, and it may be involved in neurotrophic support by BDNF.

Cell-autonomous and differential endocannabinoid signaling impacts the development of presynaptic retinal ganglion cell axon connectivity in vivo.

Cannabis exposure during gestation evokes significant molecular modifications to neurodevelopmental programs leading to neurophysiological and behavioral abnormalities in humans. The main neuronal receptor for Δ9-tetrahydrocannabinol (THC) is the type-1 cannabinoid receptor CB1R, one of the most abundant G-protein-coupled receptors in the nervous system. While THC is the major psychoactive phytocannabinoid, endocannabinoids (eCBs) are the endogenous ligands of CB1R and are known to act as retrograde messengers to modulate synaptic plasticity at different time scales in the adult brain. Accumulating evidence indicates that eCB signaling through activation of CB1R plays a central role in neural development. During development, most CB1R localized to axons of projection neurons, and in mice eCB signaling impacts axon fasciculation. Understanding of eCB-mediated structural plasticity during development, however, requires the identification of the precise spatial and temporal dynamics of CB1R-mediated modifications at the level of individual neurons in the intact brain. Here, the cell-autonomous role of CB1R and the effects of CB1R-mediated eCB signaling were investigated using targeted single-cell knockdown and pharmacologic treatments in Xenopus. We imaged axonal arbors of retinal ganglion cells (RGCs) in real time following downregulation of CB1R via morpholino (MO) knockdown. We also analyzed RGC axons with altered eCB signaling following treatment with URB597, a selective inhibitor of the enzyme that degrades Anandamide (AEA), or JZL184, an inhibitor of the enzyme that blocks 2-Arachidonoylglycerol (2-AG) hydrolysis, at two distinct stages of retinotectal development. Our results demonstrate that CB1R knockdown impacts RGC axon branching at their target and that differential 2-AG and AEA-mediated eCB signaling contributes to presynaptic structural connectivity at the time that axons terminate and when retinotectal synaptic connections are made. Altering CB1R levels through CB1R MO knockdown similarly impacted dendritic morphology of tectal neurons, thus supporting both pre- and postsynaptic cell-autonomous roles for CB1R-mediated eCB signaling.

DSCAM is differentially patterned along the optic axon pathway in the developing Xenopus visual system and guides axon termination at the target.

BACKGROUND: The Xenopus retinotectal circuit is organized topographically, where the dorsal-ventral axis of the retina maps respectively on to the ventral-dorsal axis of the tectum; axons from the nasal-temporal axis of the retina project respectively to the caudal-rostral axis of the tectum. Studies throughout the last two decades have shown that mechanisms involving molecular recognition of proper termination domains are at work guiding topographic organization. Such studies have shown that graded distribution of molecular cues is important for topographic mapping. However, the complement of molecular cues organizing topography along the developing optic nerve, and as retinal axons cross the chiasm and navigate towards and innervate their target in the tectum, remains unknown. Down syndrome cell adhesion molecule (DSCAM) has been characterized as a key molecule in axon guidance, making it a strong candidate involved in the topographic organization of retinal fibers along the optic path and at their target. METHODS: Using a combination of whole-brain clearing and immunohistochemistry staining techniques we characterized DSCAM expression and the projection of ventral and dorsal retinal fibers starting from the eye, following to the optic nerve and chiasm, and into the terminal target in the optic tectum in Xenopus laevis tadpoles. We then assessed the effects of DSCAM on the establishment of retinotopic maps through spatially and temporally targeted DSCAM knockdown on retinal ganglion cells (RGCs) with axons innervating the optic tectum. RESULTS: Highest expression of DSCAM was localized to the ventral posterior region of the optic nerve and chiasm; this expression pattern coincides with ventral fibers derived from ventral RGCs. Targeted downregulation of DSCAM expression on ventral RGCs affected the segregation of medial axon fibers from their dorsal counterparts within the tectal neuropil, indicating that DSCAM plays a role in retinotopic organization. CONCLUSION: These findings together with previous studies demonstrating cell-autonomous roles for DSCAM during the development of pre- and postsynaptic arbors in the Xenopus retinotectal circuit indicates that DSCAM exerts multiple roles in coordinating axon targeting and structural connectivity in the developing vertebrate visual system.

Netrin participates in the development of retinotectal synaptic connectivity by modulating axon arborization and synapse formation in the developing brain.

Netrin has been implicated in retinal ganglion cell (RGC) axon pathfinding in a number of species. In Xenopus laevis, RGC axons reaching their target in the optic tectum can be repelled by a netrin-1 gradient in vitro, suggesting that netrin may also function in wiring events that follow successful axon pathfinding. Here, we examined the contribution of netrin to RGC axon arborization and synapse formation at the target. Time-lapse confocal microscopy imaging of individual RGC axons coexpressing GFP-synaptobrevin and DsRed in the intact Xenopus brain demonstrated a role for deleted in colorectal cancer (DCC)-mediated netrin signaling. Microinjection of netrin-1 into the tectum induced a rapid and transient increase in presynaptic site addition that resulted in higher presynaptic site density over a 24 h observation period. Moreover, netrin induced dynamic axon branching, increasing branch addition and retraction; a behavior that ultimately increased total branch number. In contrast, microinjection of DCC function-blocking antibodies prevented the increase in presynaptic site number normally observed in control axons as well as the associated increase in branch number and axon arbor growth. Dynamic analysis of axon arbors demonstrated that the effects of anti-DCC on axon morphology and presynaptic connectivity were attributable to a specific decrease in new synapse and branch additions, without affecting the stability of existing synapses and branches. Together, these results indicate that, in the absence of DCC signaling, RGC axons fail to branch and differentiate, and support a novel role for netrin in later phases of retinotectal development.

Imaging the Dynamic Branching and Synaptic Differentiation of Xenopus Optic Axons In Vivo.

In the developing Xenopus tadpole visual system, the targeting and branching of optic axons in the brain is a dynamic process that is closely intertwined with the morphological differentiation and maturation of their postsynaptic neurons and with the formation, stabilization, and elimination of functional synapses. The coordinated addition and retraction of axonal and dendritic branches guides the gradual recognition between pre- and postsynaptic neuronal partners, which subsequently allows synaptic connections to be formed. Axon and dendrite branching and selective synapse formation and stabilization are developmental mechanisms largely orchestrated by an array of signaling molecules that interact in vivo for the proper formation of functional visual circuits. In vivo real-time imaging of individual fluorophore-labeled neurons in living Xenopus tadpoles has allowed investigation of molecular and cellular mechanisms mediating circuit assembly at a cellular level in the intact organism. In this protocol, we describe the use of bulk and single-cell electroporation to rapidly and efficiently transfect individual retinal ganglion cells (RGCs) with different reagents and to simultaneously visualize optic axon arbor morphology and presynaptic sites in real time. Similar techniques for labeling and visualizing RGC axons can be combined with the use of morpholino antisense oligonucleotides, as we describe here, to alter gene expression cell autonomously.

Cell-autonomous TrkB signaling in presynaptic retinal ganglion cells mediates axon arbor growth and synapse maturation during the establishment of retinotectal synaptic connectivity.

BDNF contributes to the activity-dependent establishment and refinement of visual connectivity. In Xenopus, BDNF applications in the optic tectum influence retinal ganglion cell (RGC) axon branching and promote synapse formation and stabilization. The expression patterns of BDNF and TrkB suggest that BDNF specifically regulates the maturation of RGC axons at the target. It is possible, however, that BDNF modulates retinotectal synaptic connectivity by differentially influencing presynaptic RGC axons and postsynaptic tectal cells. Here, we combined single-cell expression of a dominant-negative TrkB-enhanced green fluorescent protein (GFP) fusion protein with confocal microscopy imaging in live Xenopus tadpoles to differentiate between presynaptic and postsynaptic actions of BDNF. Disruption of TrkB signaling in individual RGCs influenced the branching and synaptic maturation of presynaptic axon arbors. Specifically, GFP-TrkB.T1 overexpression increased the proportion of axons with immature, growth cone-like morphology, decreased axon branch stability, and increased axon arbor degeneration. In addition, GFP-TrkB.T1 overexpression reduced the number of red fluorescent protein-synaptobrevin-labeled presynaptic specializations per axon terminal. In contrast, overexpression of GFP-TrkB.T1 in tectal neurons did not alter synaptic number or the morphology or dynamic behavior of their dendritic arbors. Electron microscopy analysis revealed a significant decrease in the number of mature synaptic profiles and in the number of docked synaptic vesicles at retinotectal synapses made by RGC axons expressing GFP-TrkB.T1. Together, our results demonstrate that presynaptic TrkB signaling in RGCs is a key determinant in the establishment of visual connectivity and indicate that changes in tectal neuron synaptic connectivity are secondary to the BDNF-elicited enhanced stability and growth of presynaptic RGCs.

DSCAM differentially modulates pre- and postsynaptic structural and functional central connectivity during visual system wiring.

BACKGROUND: Proper patterning of dendritic and axonal arbors is a critical step in the formation of functional neuronal circuits. Developing circuits rely on an array of molecular cues to shape arbor morphology, but the underlying mechanisms guiding the structural formation and interconnectivity of pre- and postsynaptic arbors in real time remain unclear. Here we explore how Down syndrome cell adhesion molecule (DSCAM) differentially shapes the dendritic morphology of central neurons and their presynaptic retinal ganglion cell (RGC) axons in the developing vertebrate visual system. METHODS: The cell-autonomous role of DSCAM, in tectal neurons and in RGCs, was examined using targeted single-cell knockdown and overexpression approaches in developing Xenopus laevis tadpoles. Axonal arbors of RGCs and dendritic arbors of tectal neurons were visualized using real-time in vivo confocal microscopy imaging over the course of 3 days. RESULTS: In the Xenopus visual system, DSCAM immunoreactivity is present in RGCs, cells in the optic tectum and the tectal neuropil at the time retinotectal synaptic connections are made. Downregulating DSCAM in tectal neurons significantly increased dendritic growth and branching rates while inducing dendrites to take on tortuous paths. Overexpression of DSCAM, in contrast, reduced dendritic branching and growth rate. Functional deficits mediated by tectal DSCAM knockdown were examined using visually guided behavioral assays in swimming tadpoles, revealing irregular behavioral responses to visual stimulus. Functional deficits in visual behavior also corresponded with changes in VGLUT/VGAT expression, markers of excitatory and inhibitory transmission, in the tectum. Conversely, single-cell DSCAM knockdown in the retina revealed that RGC axon arborization at the target is influenced by DSCAM, where axons grew at a slower rate and remained relatively simple. In the retina, dendritic arbors of RGCs were not affected by the reduction of DSCAM expression. CONCLUSIONS: Together, our observations implicate DSCAM in the control of both pre- and postsynaptic structural and functional connectivity in the developing retinotectal circuit, where it primarily acts as a neuronal brake to limit and guide postsynaptic dendrite growth of tectal neurons while it also facilitates arborization of presynaptic RGC axons cell autonomously.

Local and target-derived brain-derived neurotrophic factor exert opposing effects on the dendritic arborization of retinal ganglion cells in vivo.

The dendritic and axonal arbors of developing retinal ganglion cells (RGCs) are exposed to two sources of BDNF: RGC dendrites are exposed to BDNF locally within the retina, and RGC axons are exposed to BDNF at the target, the optic tectum. Our previous studies demonstrated that increasing tectal BDNF levels promotes RGC axon terminal arborization, whereas increasing retinal BDNF levels inhibits RGC dendritic arborization. These results suggested that differential neurotrophic action at the axon versus dendrite might be responsible for the opposing effects of BDNF on RGC axonal versus dendritic arborization. To explore this possibility, we examined the effects of altering BDNF levels at the optic tectum on the elaboration of RGC dendritic arbors in the retina. Increasing tectal BDNF levels resulted in a significant increase in dendritic branching, whereas neutralizing endogenous tectal BDNF with function-blocking antibodies significantly decreased dendritic arbor complexity. Thus, RGC dendritic arbors react in opposing manners to retinal- versus tectal-derived BDNF. Alterations in retinal BDNF levels, however, did not affect axon terminal arborization. Thus, RGC dendritic arborization is controlled in a complementary manner by both local and target-derived sources of BDNF, whereas axon arborization is modulated solely by neurotrophic interactions at the target. Together, our results indicate that developing RGCs modulate dendritic arborization by integrating signals from discrete sources of BDNF in the eye and brain. Differential integration of spatially discrete neurotrophin signals within a single neuron may therefore finely tune afferent and efferent neuronal connectivity.

Neurotrophic regulation of retinal ganglion cell synaptic connectivity: from axons and dendrites to synapses.

This review highlights important events during the morphological development of retinal ganglion cells (RGCs), focusing on mechanisms that control axon and dendritic arborization as a means to understand synaptic connectivity with special emphasis on the role of neurotrophins during structural and functional development of RGCs. Neurotrophins and their receptors participate in the development of visual connectivity at multiple levels. In the visual system, neurotrophins have been shown to exert various developmental influences, from guiding the morphological differentiation of neurons to controlling the functional plasticity of visual circuits. This review article examines the role of neurotrophins, and in particular of BDNF, during the morphological development of RGCs, and discusses potential interactions between activity and neurotrophins during development of neuronal connectivity.

Netrin-1 directs dendritic growth and connectivity of vertebrate central neurons in vivo.

BACKGROUND: Netrins are a family of extracellular proteins that function as chemotropic guidance cues for migrating cells and axons during neural development. In the visual system, netrin-1 has been shown to play a key role in retinal ganglion cell (RGC) axon growth and branching at the target, where presynaptic RGC axons form partnerships with the dendrites of tectal neurons. However, the signals that guide the connections between RGC axons and their postsynaptic partners are yet unknown. Here, we explored dynamic cellular mechanisms by which netrin-1 influences visual circuit formation, particularly those that impact postsynaptic neuronal morphology and connectivity during retinotectal wiring. RESULTS: Time-lapse in vivo imaging of individual Xenopus laevis optic tectal neurons co-expressing tdTomato and PSD95-GFP revealed rapid remodeling and reorganization of dendritic arbors following acute manipulations in netrin-1 levels. Effects of altered netrin signaling on developing dendritic arbors of tectal neurons were distinct from its effects on presynaptic RGC axons. Within 4 h of treatment, tectal injection of recombinant netrin-1 or sequestration of endogenous netrin with an UNC-5 receptor ectodomain induced significant changes in the directionality and orientation of dendrite growth and in the maintenance of already established dendrites, demonstrating that relative levels of netrin are important for these functions. In contrast, altering DCC-mediated netrin signaling with function-blocking antibodies induced postsynaptic specialization remodeling and changed growth directionality of already established dendrites. Reducing netrin signaling also decreased avoidance behavior in a visually guided task, suggesting that netrin is essential for emergent visual system function. CONCLUSIONS: These in vivo findings together with the patterns of expression of netrin and its receptors reveal an important role for netrin in the early growth and guidance of vertebrate central neuron dendritic arbors. Collectively, our studies indicate that netrin shapes both pre- and postsynaptic arbor morphology directly and in multiple ways at stages critical for functional visual system development.

Embryonic GABAergic spinal commissural neurons project rostrally to mesencephalic targets.

Although spinal commissural neurons serve as a model system for studying the mechanisms that underlie axonal pathfinding during development, little is known about their synaptic targets. Previously we identified a group of ventromedially located commissural neurons in rat spinal cord that are gamma-aminobutyric acid (GABA)-ergic and express L1 CAM on their axons. In this study, serial sagittal sections of embryos (E12-15) were processed for glutamic acid decarboxylase (GAD)-65 and L1 immunocytochemistry and showed labeled commissural axons coursing rostrally within the ventral marginal zone. Both GAD65- and L1-positive axons extended rostrally out of the spinal cord into the central part of the medulla and then into the midbrain. GAD65-positive axons branched and ended abruptly within the lateral midbrain. To determine the targets of these ventral commissural neurons, embryos (E13-15) were injected with DiI into the ventromedial spinal cord. At all three ages, DiI-labeled axons projected rostrally in the contralateral ventral marginal zone, turned into the central medulla, and then traveled to the midbrain. DiI-labeled axons appeared to terminate in the lateral midbrain by branching into small, punctate structures. In reciprocal experiments, DiI injected into the lateral midbrain identified an axon pathway that coursed through the brainstem, into the spinal cord ventral marginal zone, and then filled cell bodies in the contralateral ventromedial spinal cord. A spatial and temporal coincidence was apparent between the GAD65/L1- and the DiI-labeled pathways. Together these findings suggest that some GABAergic commissural neurons are early projection neurons to midbrain targets and most likely represent a spinomesencephalic pathway to the midbrain reticular formation.

Dynamic responses of Xenopus retinal ganglion cell axon growth cones to netrin-1 as they innervate their in vivo target.

Netrin-1 influences retinal ganglion cell (RGC) axon pathfinding and also participates in the branching and synaptic differentiation of mature RGC axons at their target. To investigate whether netrin also serves as an early target recognition signal in the brain, we examined the dynamic behavior of Xenopus RGC axons soon after they innervate the optic tectum. Time-lapse confocal microscopy imaging of RGC axons expressing enhanced yellow fluorescent protein demonstrated that netrin-1 is involved in early axon branching, as recombinant netrin-1 halted further advancement of growth cones into the tectum and induced back branching. RGC growth cones exhibited differential responses to netrin-1 that depended on the degree of differentiation of the axon and the developmental stage of the tadpole. Netrin-1 decreased the total number of branches on newly arrived RGC growth cones at the target, but increased the dynamic branching of more mature arbors at the later developmental stage. To further explore the response of axonal growth cones to netrin, Xenopus RGC axons were followed in culture by time-lapse imaging. Exposure to netrin-1 rapidly increased the forward advancement of the axon and decreased the size and expanse of the growth cone, while also inducing back branching. Taken together, the differential in vivo and in vitro responses to netrin-1 suggest that netrin alone is not sufficient to induce the cessation of growth cone advancement in the absence of a target but can independently modulate axon branching. Collectively, our findings reveal a novel role for netrin on RGC axon branch initiation as growth cones innervate their target.

Cell-autonomous alterations in dendritic arbor morphology and connectivity induced by overexpression of MeCP2 in Xenopus central neurons in vivo.

Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Mutations in the MeCP2 gene have been linked to Rett syndrome, a severe X-linked progressive neurodevelopmental disorder, and one of the most common causes of mental retardation in females. MeCP2 duplication and triplication have also been found to affect brain development, indicating that both loss of function and gain in MeCP2 dosage lead to similar neurological phenotypes. Here, we used the Xenopus laevis visual system as an in vivo model to examine the consequence of increased MeCP2 expression during the morphological maturation of individual central neurons in an otherwise intact brain. Single-cell overexpression of wild-type human MeCP2 was combined with time-lapse confocal microscopy imaging to study dynamic mechanisms by which MeCP2 influences tectal neuron dendritic arborization. Analysis of neurons co-expressing DsRed2 demonstrates that MeCP2 overexpression specifically interfered with dendritic elaboration, decreasing the rates of branch addition and elimination over a 48 hour observation period. Moreover, dynamic analysis of neurons co-expressing wt-hMeCP2 and PSD95-GFP revealed that even though neurons expressing wt-hMeCP2 possessed significantly fewer dendrites and simpler morphologies than control neurons at the same developmental stage, postsynaptic site density in wt-hMeCP2-expressing neurons was similar to controls and increased at a rate higher than controls. Together, our in vivo studies support an early, cell-autonomous role for MeCP2 during the morphological differentiation of neurons and indicate that perturbations in MeCP2 gene dosage result in deficits in dendritic arborization that can be compensated, at least in part, by synaptic connectivity changes.

Synaptic maturation of the Xenopus retinotectal system: effects of brain-derived neurotrophic factor on synapse ultrastructure.

Synaptogenesis is a dynamic process that involves structural changes in developing axons and dendrites as synapses form and mature. The visual system of Xenopus laevis has been used as a model to study dynamic changes in axons and dendrites as synapses form in the living brain and the molecular mechanisms that control these processes. Brain-derived neurotrophic factor (BDNF) contributes to the establishment and refinement of visual connectivity by modulating retinal ganglion cell (RGC) axon arborization and presynaptic differentiation. Here, we have analyzed the ultrastructural organization of the Xenopus retinotectal system to understand better the maturation of this synaptic circuit and the relation between synapse ultrastructure and the structural changes in connectivity that take place in response to BDNF. Expression of yellow fluorescent protein (YFP) followed by preembedding immunoelectron microscopy was used to identify RGC axons specifically in living tadpoles. Injection of recombinant BDNF was used to alter endogenous BDNF levels acutely in the optic tectum. Our studies reveal a rapid transition from a relatively immature synaptic circuit in which retinotectal synapses are formed on developing filopodial-like processes to a circuit in which RGC axon terminals establish synapses with dendritic shafts and spines. Moreover, our studies reveal that BDNF treatment increases the number of spine synapses and docked vesicle number at YFP-identified synaptic sites within 24 hours of treatment. These fine structural changes at retinotectal synapses are consistent with the role that BDNF plays in the functional maturation of synaptic circuits and with dynamic, rapid changes in synaptic connectivity during development.

Brain-derived neurotrophic factor and the development of structural neuronal connectivity.

During development, neural networks are established in a highly organized manner, which persists throughout life. Neurotrophins play crucial roles in the developing nervous system. Among the neurotrophins, brain-derived neurotrophic factor (BDNF) is highly conserved in gene structure and function during vertebrate evolution, and serves an important role during brain development and in synaptic plasticity. BDNF participates in the formation of appropriate synaptic connections in the brain, and disruptions in this process contribute to disorders of cognitive function. In this review, we first briefly highlight current knowledge on the expression, regulation, and secretion of BDNF. Further, we provide an overview of the possible actions of BDNF in the development of neural circuits, with an emphasis on presynaptic actions of BDNF during the structural development of central neurons.

Spatially patterned gradients of synaptic connectivity are established early in the developing retina.

Retinal neurons receive input from other cells via synapses and the position of these synapses on the neurons reflects the retinal regions from which information is received. A new study in Neural Development establishes that the spatial distribution of excitatory synaptic inputs emerges at the onset of synapse formation rather than as a result of changes during neuronal reorganisation.

Imaging retinotectal synaptic connectivity.

INTRODUCTIONThis protocol describes a method for imaging synaptic sites in individual axon or dendritic terminals in live, developing embryos. By selectively targeting expression of green fluorescent protein (GFP)-tagged pre- and post-synaptic proteins to developing neurons in vivo, it is possible to visualize synaptic sites and to correlate their distribution and dynamics with changes in the morphology of axon or dendritic arbors labeled with a red fluorescent molecule. The techniques described here may be applied to almost any neuronal circuit or neuron type, as long as the embryonic tissues are accessible for transfection and the neurons of interest are located at a depth that is within the working distance of the objective. These techniques have been optimized for Xenopus embryos, but they are applicable to other vertebrate systems such as zebrafish and chick embryos.

BDNF increases synapse density in dendrites of developing tectal neurons in vivo.

Neuronal connections are established through a series of developmental events that involve close communication between pre- and postsynaptic neurons. In the visual system, BDNF modulates the development of neuronal connectivity by influencing presynaptic retinal ganglion cell (RGC) axons. Increasing BDNF levels in the optic tectum of Xenopus tadpoles significantly increases both axon arborization and synapse density per axon terminal within a few hours of treatment. Here, we have further explored the mechanisms by which BDNF shapes synaptic connectivity by imaging tectal neurons, the postsynaptic partners of RGCs. Individual neurons were co-labeled with DsRed2 and a GFP-tagged postsynaptic density protein (PSD95-GFP) to visualize dendritic morphology and postsynaptic specializations simultaneously in vivo. Immunoelectron microscopy confirmed that PSD95-GFP predominantly localized to ultrastructurally identified synapses. Time-lapse confocal microscopy of individual, double-labeled neurons revealed a coincident, activity-dependent mechanism of synaptogenesis and axon and dendritic arbor growth, which is differentially modulated by BDNF. Microinjection of BDNF into the optic tectum significantly increased synapse number in tectal neuron dendritic arbors within 24 hours, without significantly influencing arbor morphology. BDNF function-blocking antibodies had opposite effects. The BDNF-elicited increase in synapse number complements the previously observed increase in presynaptic sites on RGC axons. These results, together with the timescale of the response by tectal neurons, suggest that the effects of BDNF on dendritic synaptic connectivity are secondary to its effects on presynaptic RGCs. Thus, BDNF influences synaptic connectivity in multiple ways: it enhances axon arbor complexity expanding the synaptic territory of the axon, while simultaneously coordinating synapse formation and stabilization with individual postsynaptic cells.

BDNF stabilizes synapses and maintains the structural complexity of optic axons in vivo.

Brain-derived neurotrophic factor (BDNF) modulates synaptic connectivity by increasing synapse number and by promoting activity-dependent axon arbor growth. Patterned neuronal activity is also thought to influence the morphological maturation of axonal arbors by directly influencing the stability of developing synapses. Here, we used in vivo time-lapse imaging to examine the relationship between synapse stabilization and axon branch stabilization, and to better understand the participation of BDNF in synaptogenesis. Green fluorescent protein (GFP)-tagged synaptobrevin II was used to visualize presynaptic specializations in individual DsRed2-labeled Xenopus retinal axons arborizing in the optic tectum. Neutralizing endogenous tectal BDNF with function-blocking antibodies significantly enhanced GFP-synaptobrevin cluster elimination, a response that was paralleled by enhanced branch elimination. Thus, synapse dismantling was associated with axon branch pruning when endogenous BDNF levels were reduced. To obtain a second measure of the role of BDNF during synapse stabilization, we injected recombinant BDNF in tadpoles with altered glutamate receptor transmission in the optic tectum. Tectal injection of the NMDA receptor antagonists APV or MK801 transiently induced GFP-synaptobrevin cluster dismantling, but did not significantly influence axon branch addition or elimination. BDNF treatment rescued synapses affected by NMDA receptor blockade: BDNF maintained GFP-synaptobrevin cluster density by maintaining their addition rate and rapidly inducing their stabilization. Consequently, BDNF influences synaptic connectivity in multiple ways, promoting not only the morphological maturation of axonal arbors, but also their stabilization, by a mechanism that influences both synapses and axon branches.