Expression of netrin-1 and its receptors in the tectum during visual circuit development

In the developing Xenopus visual system, RGC axons at their target express DCC and differentially respond to netrin-1 depending on their maturational state by halting growth cone advancement within the target [12] or by rapidly increasing the number of green fluorescent protein (GFP)-tagged presynaptic specializations and subsequently increasing branch number [11]. To further characterize the roles of netrin-1 during visual circuit development, we examined the expression of netrin-1 and its receptors DCC and UNC-5 in the optic tectum at the time when tectal neurons differentiate and form connections with branching RGC axons (Fig. 1a). Quantitative reverse transcription polymerase chain reaction (RT-PCR) showed DCC, UNC-5, and netrin-1 mRNA expression in the midbrain of stage 41 to 45 tadpoles (not shown). In situ hybridization studies revealed that netrin-1 mRNA is expressed in the midbrain of stage 45 tadpoles predominantly near the ventricle wall, in a ventral-high to dorsal-low gradient (Fig. 1b, c). Immunostaining with antibodies to UNC-5 and to DCC demonstrated areas of overlapping expression for these two netrin-1 receptors within the midbrain at this same stage (Fig. 1d–g). In the optic tectum, UNC-5 immunoreactivity was restricted primarily to cell bodies and proximal processes in the dorso-caudal midbrain (Figs. 1e–i, 2c) and was absent from the tectal neuropil where presynaptic retinal ganglion cell (RGC) axons terminate (Fig. 1h, i). Immunostaining with an antibody against the extracellular domain of DCC revealed that DCC was localized throughout the tectal neuropil (Fig. 1d–g, Fig. 2d), consistent with findings using antibodies that recognize the intracellular domain of DCC [11]. Moreover, DCC immunoreactivity was found around tectal cell bodies and in neuronal processes that extended to the tectal neuropil where primary dendrites begin to branch. Defined patterns of UNC-5 and DCC expression were also found in the forebrain, pre-tectum, caudal tectum, hindbrain, and spinal cord (Fig. 2). Consequently, the patterns of netrin-1 mRNA (Fig. 1b, c) and protein expression [11] and the localization of DCC and UNC-5 receptors within the optic tectum suggest that tectal neurons can respond to netrin-1 directly.

Fig. 1 Expression of netrin-1 and of its receptors DCC and UNC-5 in stage 45 Xenopus optic tectum. a Schematic of coronal section of Xenopus retinotectal circuit. RGC axons (green) travel from the contralateral eye to connect with tectal neurons in the neuropil (blue). b, c In situ hybridization with Xenopus-specific antisense netrin-1 probes. Coronal sections of the midbrain at the level of the optic tectum show ventral-high (double arrows) to dorsal-low (arrow) netrin-1 mRNA expression along the ventricle wall. d–g Coronal and h, i horizontal sections show DCC and UNC-5 expression. d–g Co-immunostaining illustrates the differential distribution of UNC-5 (red) and DCC (green) immunoreactivity. d DCC immunoreactivity (green) is localized to the cell bodies in the dorsal tectum and proximal dendrites and to incoming axons near the dorsal neuropil (arrow). The tectal neuropil (np) is also positive for DCC. The low- (e, f) and high- (g) magnification coronal images show UNC-5 (red) and DCC (green) co-localization, with UNC-5 being localized to a subset of cells that also expresses DCC (g, arrowheads). f Counterstaining with DAPI (blue) serves to distinguish nuclear staining from cytoplasmic UNC-5 (red) and DCC (green) expression in tectal cells. h UNC-5 immunoreactivity (green) is localized to a subset of cell bodies in the dorsal area of the tectum and area adjacent to the tectal neuropil identified by immunostaining with antibodies to the presynaptic protein SNAP-25 (red). i Anterograde labeling with rhodamine dextran shows that RGC axons (red) terminate in the areas of the tectal neuropil (arrow) where UNC-5 immunopositive neurons localize (green). D dorsal, V ventral, C caudal, R rostral, L lateral, np neuropil. Scale bars: 50 μm in b–f, 20 μm in g, 20 μm in h–i Full size image

Fig. 2 Specific patterns of DCC and UNC-5 expression in the X. laevis central nervous system. Immunostaining with antibodies to UNC-5 (red) and DCC (green) revealed specific patterns of expression of the netrin-1 receptors in stage 45 tadpoles. a–g UNC-5 (red) and DCC (green) immunoreactivity in the forebrain (a), pre-tectum (b), caudal tectum (e), hindbrain (f), and rostral spinal cord (g) demonstrate a specific pattern of expression for each of these receptors within subpopulations of neurons in the central nervous system. c UNC-5 immunostaining (red) localizes to subpopulations of neurons in the dorsal tectum, lateral-ventral midbrain, ventral midline (vm), and infundibulum (if). d DCC immunoreactivity (green) is localized in dorsal tectal neuron cell bodies and processes in the tectum and ventral midline, as well as in the tectal neuropil (np). e, f Note the specificity of immunostaining and co-localization of UNC-5 and DCC expression in subpopulations of cells in the caudal tectum (e) and hindbrain (f) and the localization of DCC receptors to discrete fiber tracts (arrows). g, h UNC-5 (red) and DCC (green) immunoreactivity in the rostral (g) and caudal (h) spinal cord is localized to fiber tracts and ventral midline in agreement with published observations in Xenopus and other species (for review, see [42, 5, 43–45]). DCC immunoreactivity in the spinal cord is similar when staining with antibodies directed against the extracellular (g) or intracellular (h, bottom) domains of DCC. Counterstaining with DAPI (blue) serves to distinguish nuclear staining from UNC-5 (red) and DCC (green) expression in cell bodies and fiber tracts. Scale bars: 50 μm Full size image

Acute manipulations in netrin levels or DCC signaling

To explore dynamic mechanisms by which netrin-1 influences postsynaptic neuronal morphology and connectivity in the retinotectal system, we altered endogenous netrin-1 levels or DCC signaling in the stage 45 tadpole optic tectum by microinjecting recombinant netrin-1, an UNC-5 receptor ectodomain that sequesters netrin (UNC5H2-Ig), or function-blocking antibodies to DCC. We examined protein distribution immediately after injection to determine rates of diffusion from the injection site (Fig. 3) as a means to evaluate the effectiveness of the acute treatments. Immunostaining with specific antibodies to netrin revealed that netrin-1 injection into the ventricle and lateral side of the optic tectum resulted in higher immunoreactivity in the neuropil near the injection site and an even distribution of the exogenous protein within the cell body layer above the endogenous netrin expression (Fig. 3b–e). Quantitative analysis of the immunofluorescent signal further demonstrated that the treatment was effective in increasing tectal netrin levels (Fig. 3c). Staining with a fluorescent antihuman IgG antibody allowed visualization of the injected UNC5H2 Fc chimeric protein and showed graded distribution of UNC5H2-Ig in the tectal hemisphere that received treatment (Fig. 3f), which could alter the localized spatial distribution of endogenous netrin-1. Similarly, immunostaining with fluorescent anti-mouse IgG to visualize the injected function-blocking antibody to DCC demonstrated that anti-DCC effectively diffused within the neuropil (Fig. 3g) and had the ability to bind the endogenous receptor and prevent signaling.

Fig. 3 Protein diffusion after treatment. a Schematic of coronal view of stage 45 Xenopus retinotectal circuit depicting injection sites (red arrows) and spread of injected proteins (violet color). b Coronal section at the level of the optic tectum immunostained with antibodies to netrin-1. Note endogenous netrin immunoreactivity in cell body layer and neuropil. c–g Sections at the level of the optic tectum of tadpoles injected with vehicle, recombinant netrin-1, UNC5H2-Ig, or anti-DCC were immunostained to examine the spread of the injected proteins after treatment. c Quantitative analysis of fluorescence intensity in sections of uninjected tadpoles (Endogenous Netrin) or tadpoles injected with recombinant netrin (Injected rNetrin-1). The relative levels of netrin within the cell body layer and the neuropil are illustrated by the average pixel intensity values along the medial-to-lateral axis of the tectum. The zero value in the X-axis corresponds to the cell body layer-neuropil boundary; negative X-coordinates represent distance from the boundary to the ventricle while positive X-coordinates represent distance from the boundary to the lateral-most neuropil. n = 10 brain sections per group, from four tadpole brains per group, with three 20-pixel-wide line scans quantified per section. Error bars represent the standard error of the mean. d–g Sample coronal sections of tadpoles injected with vehicle (d), recombinant netrin-1 (e), UNC5H2-Ig (f), or anti-DCC (g) immunostained with chick antibodies to netrin-1 and Alexa 488 secondary antibodies to chick IgG (top; d, e) or stained with Alexa 488 secondary antibodies to human IgG (top; f) or mouse IgG (top; g). The pseudo-color images in d–g (bottom) show the relative intensity of the Alexa fluor 488 fluorescence. Pixel intensity values ranged from 0 (black) to 255 (white) as illustrated by the color-scale bar (d, bottom). Note the increased immunofluorescence in the cell body layer and neuropil of netrin-1-treated tadpoles (e) when compared to vehicle-injected controls (d) and with endogenous netrin-1 expression (b). In f and g, the relatively higher fluorescence intensity in the hemisphere that received the injection (red arrows) and the diffusion patterns of the proteins are more evident in the pseudo-color images. In g, white arrows point to fluorescently labeled cells in the injected tectal hemisphere. Scale bars in b, d–g: 50 μm Full size image

Netrin differentially affects retinal ganglion cell axons and tectal neuron dendrites

To examine if netrin-1 shapes postsynaptic neuronal connectivity in addition to influencing RGCs, we imaged pairs of fluorescently labeled pre- and postsynaptic arbors branching in the optic tectum of stage 45 tadpoles. The simultaneous, dynamic behavior of individual tectal neurons expressing tdTomato and of RGC axons expressing GFP was followed in vivo by confocal microscopy (Fig. 4a). In control tadpoles, both presynaptic and postsynaptic arbors gradually grew towards one another within the tectal neuropil (Fig. 4b). Upon acute injection of recombinant netrin-1, however, tectal neurons showed rapid reorganization of their dendritic arbor (Fig. 4c) while RGC axons continued to grow forward and elaborate. Dendrites of tectal neurons appeared to alter their branch directionality away from the neuropil and from branching RGC axons (Fig. 4c, insets). As tectal neurons responded to recombinant netrin-1 by remodeling their dendritic arbors, RGC axons increased their number of branches significantly more than controls 24 h after netrin-1 treatment (control 170.5 ± 13.79 % n = 4, netrin 247.8 ± 15.93 % n = 4, p = 0.0105; not shown graphically) in agreement with previous findings [11].

Fig. 4 Rapid remodeling of dendritic arbors upon acute manipulations in netrin signaling. a Schematic diagram of a stage 45 Xenopus tectal midbrain (horizontal view). Tectal neurons (red) make dendritic connections with contralateral RGC axons (green) within the tectal neuropil. b, c Sample RGC axons and tectal neurons, visualized by expression of GFP and tdTomato, respectively, in control (b) and netrin-treated (c) tadpoles. Note change in tectal neuron dendritic architecture evident at 4 and 24 h after netrin-1 treatment (inserts). d–g Confocal projections of representative tectal neurons co-expressing tdTomato (red) and PSD95-GFP (green) in tadpoles injected with control vehicle solution (d), Netrin (e), UNC5H2-Ig (f), or Netrin + UNC5H2-Ig (g). Note the emergence of an alternative primary dendrite (arrow) growing towards the midline in neurons exposed to netrin-1 or UNC5H2-Ig. Tadpoles treated with netrin + UNC5H2-Ig appeared identical to controls. Axons of tectal neurons are labeled by the asterisks. Scale bars: 20 μm Full size image

Effects of netrin-1 on the morphological development of developing tectal neurons

To further characterize the differential response of tectal neurons to netrin-1, we imaged individual neurons co-expressing tdTomato and PSD95-GFP before (time 0), 2, 4, and 24 h after netrin-1 treatment. Control neurons extended their dendritic arbor without altering their basic architecture (Fig. 4b, d). In contrast, neurons in tadpoles treated with netrin-1 rapidly reorganized their dendritic arbors (Fig. 4c, e). Quantitative analysis of dendrite branching showed that treatment with exogenous netrin-1 did not significantly influence total branch number or dendritic arbor length of tectal neurons (Fig. 5a, b). One possibility that could account for the effects of acute netrin-1 treatment on dendritic arbor shape is that activation of netrin signaling increased the exploratory activity of dendritic processes which leads to a dynamic reorganization of the arbor without affecting overall branch growth. To further explore the effects of netrin, we decreased endogenous netrin levels in the tectum by injecting UNC-5 receptor bodies (UNC5H2-Ig) as a means to sequester bioavailable netrin-1 [16]. Injection of UNC5H2-Ig into the midbrain ventricle and the lateral side of the tectum also caused rapid reorganization and reorientation of tectal neuron dendritic arbors (Fig. 4f). Moreover, UNC5H2-Ig treatment significantly decreased total branch number and dendrite arbor length by 2 h, an effect that was maintained 4 h after treatment (Fig. 5a, b). Consequently, tectal neurons responded to decreased tectal netrin levels more robustly but similarly to exogenous netrin-1, suggesting that the destabilization and reorientation of dendrites may be attributed to the disruption of differential endogenous netrin expression or signaling. To further test for specificity of effects, we co-injected tadpoles with a mix of netrin-1 and UNC5H2-Ig at a ratio in which recombinant netrin-1 would neutralize the UNC-5 ectodomain dimer (1.7:2 mol:mol solution). In contrast to netrin-1 treatment alone or UNC5H2-Ig treatment alone, neurons in tadpoles co-treated with netrin and UNC5H2-Ig had morphologies and total branch number and length indistinguishable from controls (Fig. 4g; Fig. 5a, b). Therefore, our studies indicate that while responses to exogenous netrin-1 and to sequestration of endogenous netrin with the UNC-5 ectodomain are similar, they are specific to each treatment.

Fig. 5 Altering endogenous netrin levels decreases dendrite branch number and total dendritic arbor length. Effects of tectal microinjection of netrin, UNC5H2-Ig, or netrin + UNC5H2-Ig on total dendrite branch number (a) and length (b). Netrin-1 and UNC5H2-Ig altered tectal neuron morphology with a different time scale. Note that exogenous netrin-1 treatment decreased dendrite arbor length at 24 h, while the UNC5H2-Ig treatment that sequesters endogenous netrin induced a transient but significant decrease in branch number at the 0- to 2- and 0- to 4-h imaging intervals when compared to all other treatments. Co-treatment with netrin + UNC5H2-Ig did not influence branch number or length. Values are expressed as percent change from the initial 0-h imaging session. Two-way ANOVA with Bonferroni multiple comparison test; *p < 0.05, **p < 0.01. Error bars indicate SEM Full size image

DCC-mediated signaling influences dendritic growth and directionality without altering total branch number or length

In Xenopus, RGC axons respond to altered DCC receptor signaling at their target by halting their presynaptic differentiation and growth [11, 12]. To determine whether the effects of altered netrin levels on tectal neurons are also mediated through its receptor DCC, we examined dynamic changes in arbor morphology of tectal neurons following injection of function-blocking antibodies to DCC. Neurons in tadpoles treated with anti-DCC rapidly remodeled their dendritic arbors and changed their morphology when compared to controls (Fig. 6a, c) similarly but less robustly than the effects of netrin-1 (Figs. 4e, 6b). As observed for neurons in tadpoles treated with netrin-1 or with UNC5H2-Ig, anti-DCC induced the formation of ectopic basal projections in tectal neurons 2 and 4 h after treatment (Fig. 6b, c (arrows), see also Fig. 4). However, in contrast to treatment with the UNC-5 ectodomain, anti-DCC treatment did not alter total dendrite branch number or total arbor length at any imaging interval (Fig. 6d, e).

Fig. 6 Blocking DCC signaling induces changes in dendritic arbor shape without altering total branch number or length. a–c Confocal projections of representative tectal neurons co-expressing tdTomato (red) and PSD95-GFP (green) in tadpoles injected with control vehicle solution (a), netrin-1 (b), or function-blocking antibodies to DCC (c). While control neurons branch, elaborate, and add PSD95-GFP puncta (a), neurons in tadpoles treated with netrin-1 undergo dynamic remodeling of existing branches (b). Short arrows point to dendrites with altered directions of growth. Neurons in tadpoles treated with anti-DCC (c) also appear to change dendritic arbor direction and form small basal projections at 2 and 4 h post-injection (long arrows). Scale bars: 20 μm. d, e Comparison of effects of netrin and anti-DCC on total branch number (d) and dendritic arbor length (e). Note that only netrin-1 treatment decreased arbor length at 24 h (e), but neither netrin nor anti-DCC affects the total number of branches (d). Two-way ANOVA with Bonferroni multiple comparison test; *p < 0.05. Error bars indicate SEM Full size image

Altering endogenous netrin signaling induces rapid remodeling of dendritic arbors

Neurons in tadpoles treated with UNC5H2-Ig responded to decreasing netrin-1 levels by altering total branch number and length early after treatment. However, treatment with netrin-1 or anti-DCC caused remodeling of dendritic arbors without influencing total branch number or length. To further characterize the differences in tectal neuron responses to altered netrin-1 levels and DCC signaling, we analyzed branch dynamics of tectal neurons imaged over the 24-h period. Detailed quantitative analysis demonstrated that tectal neurons responded to netrin-1 and to UNC5H2-Ig through similar dynamic reorganization of their dendritic arbors. Neurons in netrin-1- and in UNC5H2-Ig-treated tadpoles increased new branch addition and decreased branch stabilization (Fig. 7a, b). Significantly more branches were added following netrin-1 or UNC5H2-Ig treatments relative to controls at all time intervals (Fig. 7a) while the stability of existing branches was also decreased (Fig. 7b). A similar shift in the distribution of neurons that responded to netrin-1 or UNC5H2-Ig with increased branch addition rates further demonstrates that neurons responded similarly to these treatments independent of their initial morphology and branch number (Fig. 7c). The rapid changes in branch addition and stability following treatment with netrin-1 alone or with UNC5H2-Ig alone therefore suggest that threshold levels of netrin protein or receptor-mediated signaling contribute to these remodeling effects. In contrast to netrin-1 and to UNC5H2-Ig, the anti-DCC treatment only induced a small but significant decrease in the stability of branches by 24 h (Fig. 7b). As for other measures, tadpoles treated with netrin and UNC5H2-Ig in combination had branch addition and branch stabilization rates similar to controls at all imaging intervals (addition 0–2 h, control 32.58 ± 2.25 %, netrin + UNC5H2-Ig 29.99 ± 3.44 %; stabilization 0–2 h, control 74.57 ± 2.45 %, netrin + UNC5H2-Ig 71.20 ± 5.72, p > 0.05 two-way ANOVA, not shown graphically), supporting the specificity of the individual treatments. Together, these results demonstrate that alterations in tectal netrin levels significantly influenced the dynamic remodeling of dendritic arbors while dendrites continued to remodel at a similar rate but failed to stabilize following blockade of DCC signaling.

Fig. 7 Acute manipulations in endogenous netrin levels induce rapid changes in dendrite remodeling. a, b Effects of netrin-1, UNC5H2-Ig, or anti-DCC treatments on new branch addition (a) and branch stabilization (b). Note that while netrin-1 and UNC5H2-Ig increased branch addition and decreased branch stabilization throughout the 24-h imaging period, the anti-DCC treatment influenced the stability of branches at the 4- to 24-h interval only. c Relative proportion of neurons with different branch addition rates. A significant shift in the distribution of neurons that responded with increased branch addition rates was observed after netrin-1 and UNC5H2-Ig treatments. Values are expressed as percent change from total branches. d Relative change in DCI values is shown for each group at all imaging intervals. Note that neurons in UNC5H2-Ig-treated tadpoles significantly decreased their complexity by 4 h compared to controls. Two-way ANOVA with Bonferroni multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate SEM Full size image

To further evaluate the morphological changes in neurons elicited by altered netrin levels and signaling, we calculated dendritic complexity index (DCI) [17], a measure of the relative proportion of primary, secondary, and higher order branches. The complexity of neurons in UNC5H2-Ig-treated tadpoles was significantly lower than controls 4 h after treatment, as shown by the relative change in DCI values between 0 and 4 h after treatment (control 2.589 ± 1.978 % vs. UNC5H2-Ig −11.760 ± 4.145 %, p < 0.01; two-way ANOVA with Bonferroni multiple comparison, Fig. 7d). We further examined whether the decrease in dendritic arbor complexity was due to changes in the addition of lower order branches or to elimination of higher order branches by quantifying the proportion of primary, secondary, tertiary, and higher order branches for each neuron. Correspondingly, the number of tertiary branches in neurons in UNC5H2-Ig-treated tadpoles was significantly lower than in controls 4 h after treatment (absolute numbers: control 5.962 ± 0.5363 vs. UNC5H2-Ig 2.933 ± 0.6053, p < 0.001; two-way ANOVA with Bonferroni multiple comparison, not shown graphically), and in proportion, tertiary branches were also lower than in controls (tertiary branches: control 35.059 ± 2.769 % vs. UNC5H2-Ig 22.995 ± 3.675 %, p < 0.05; primary branches: control 11.258 ± 1.355 % vs. UNC5H2-Ig 23.076 ± 6.477 % p < 0.05; two-way ANOVA with Bonferroni multiple comparison, not shown graphically). Neurons from tadpoles treated with anti-DCC also had a significantly lower number and proportion of tertiary branches relative to controls at 24 h (tertiary branches: control 6.389 ± 0.805, anti-DCC 3.000 ± 0.768, p < 0.001; control 34.092 ± 2.78 %, anti-DCC 20.860 ± 4.00 %, p < 0.01; two-way ANOVA with Bonferroni multiple comparison, not shown graphically). The change in number and proportion of tertiary branches in neurons in anti-DCC-treated tadpoles is consistent with the time when stable branches were also significantly decreased, although the DCI values did not differ significantly in this group from that of controls. Consequently, the changes in the dendritic arbor complexity and pruning of higher order branches reflect the active remodeling of the dendritic arbors in response to decreased netrin levels or DCC signaling.

Netrin influences the dynamics and maintenance of postsynaptic specializations

In vivo imaging studies in Xenopus and in zebrafish have shown coordinated dynamic remodeling of synapses and dendritic arbor structure during tectal neuron development [18, 19]. In control neurons co-expressing tdTomato and PSD95-GFP, new PSD95-GFP postsynaptic specializations are added and stabilized within every 2 h of imaging (Fig. 8a; see also [19]). Consistent with the increased dendrite remodeling induced by netrin-1, in vivo imaging revealed that more PSD95-GFP-labeled postsynaptic specializations were added within the first observation interval in comparison to controls (0–2 h; Fig. 8a, b, Fig. 9a). Additionally, in netrin-treated tadpoles, relatively fewer postsynaptic specializations were stabilized 4 h following treatment when compared to controls (2- to 4-h interval; Figs. 8b, 9b). Treatment with UNC5H2-Ig did not significantly alter PSD95-GFP puncta addition or stabilization at any of the observation intervals although postsynaptic specializations tended to be less stable as more branches were eliminated after UNC5H2-Ig treatment (Figs. 8c, 9b). Surprisingly, even though dendrite remodeling occurred at the same rate as controls following anti-DCC treatment (Fig. 7 above), relatively more PSD95-GFP puncta were added during the first 2-h observation interval and fewer were stabilized between 2–4 h (Figs. 8d, 9a, b), similar to the effects of netrin-1.

Fig. 8 Altered netrin-1 levels and DCC signaling impact postsynaptic cluster remodeling. a–d Confocal projections of single branches from representative tectal neurons co-expressing tdTomato (red) and PSD95-GFP (green) from control (a), netrin (b), UNC5H2-Ig (c), or Anti-DCC (d) groups before and after treatment. Dynamic remodeling of postsynaptic specializations is illustrated by the addition (green arrowheads) and elimination (yellow arrowheads) of PSD95-GFP clusters. Blue arrowheads denote puncta that remained stable from one observation interval to the next; white arrowheads denote puncta that were present at the initial observation time point but were eliminated (yellow) at 2 h. Scale bar: 20 μm Full size image

Fig. 9 Postsynaptic cluster addition and stabilization are modulated by alterations in netrin signaling. a, b Effects of netrin-1, UNC5H2-Ig, or anti-DCC treatments on postsynaptic cluster remodeling were quantified as the proportion of PSD95-GFP puncta that were added (a) and remained stable (b) within the 0–2 and 2–4 observation intervals. Note that significantly more PSD95-GFP puncta were between 0 and 2 h (a), while fewer were stable between 2 and 4 h (b) following netrin-1 or anti-DCC treatment when compared to controls. c To determine the relative stability of newly added postsynaptic clusters, we quantified relative proportion of PSD95-GFP puncta added over the 0- to 2-h interval that were lost in the subsequent 2- to 4-h interval for a subset of randomly selected neurons for each group (n = 4). PSD95-GFP puncta added from 0 to 2 h were significantly less stable in the netrin-1- or anti-DCC-treated neurons. Statistical significance was by one-way ANOVA and with unpaired t-tests. Significance when compared to control is *p < 0.05, **p < 0.01. Error bars indicate SEM Full size image

To determine if PSD95-GFP puncta newly added in response to netrin-1 or anti-DCC treatment were more likely to be destabilized and eliminated, we then analyzed a subset of neurons to determine the fate of each individual puncta 4 h after treatment (arrows, Fig. 8). New puncta added from 0–2 h were significantly more likely to be eliminated at the 2- to 4-h interval following netrin-1 or anti-DCC treatment (control 11.67 ± 7.39 % n = 4; netrin 48.98 ± 12.36 % n = 4; anti-DCC 66.47 ± 12.33 % n = 4; Fig. 9c), indicating that active postsynaptic site remodeling accompanied dendrite branch remodeling. Even though manipulations in netrin levels and in DCC signaling significantly influenced postsynaptic specialization dynamics (increased addition followed by decreased stabilization), the density of PSD95-GFP puncta was not significantly different from controls at any of the observation time points in neurons from netrin-1-, anti-DCC-, or UNC5H2-Ig-treated tadpoles (i.e., at 0–4 h; control 131.2 ± 18.57 %, netrin 89.47 ± 6.823 %; UNC5H2-Ig 132.2 ± 13.98 %; anti-DCC 152.2 ± 36.10, p = 0.2537; one-way ANOVA, Dunnett’s multiple comparison test, not shown graphically).

Manipulations in netrin signaling impact dendritic arbor directionality in multiple ways

Neurons in the optic tectum grow apical dendrites towards the tectal neuropil where they normally partner with RGC axons (Fig. 4a, b, d, and Fig. 6a). In vivo imaging showed that following netrin-1 treatment tectal neurons extended new ectopic basal projections, including a potential alternative primary dendrite (identified by the accumulation of PSD95-GFP, Fig. 4e, arrow) towards the ventricle midline while pruning or redirecting branches that normally grow towards the neuropil (Fig. 4e). Overlays of color-coded tracings (wireframes) of sample neurons imaged at 0, 2, and 4 h, as well as cumulative wireframes of a subset of neurons from each group, further illustrate the emergence of ectopic projections and dynamic changes in dendritic arbor growth in response to netrin-1, UNC5H2-Ig, or anti-DCC treatment (Fig. 10). The number of neurons that extended an alternative ectopic projection was significantly higher in netrin-treated tadpoles than in controls (control 8.33 %, netrin 42.11 %, p = 0.0131; Fisher’s exact test; Fig. 11a). Similar to netrin-1, either sequestering endogenous netrin with UNC5H2-Ig or altering DCC-mediated netrin signaling with anti-DCC resulted in a higher proportion of neurons that extended an ectopic projection away from the neuropil (control 8.33 %, anti-DCC 40.00 %, UNC5H2-Ig 40.00 %, p = 0.0370, Fisher’s exact test). To further evaluate changes in the orientation of the dendritic arbor, we calculated the vector angle for each neuron before and after treatment (Fig. 11b, see the “Methods” section). In the presence of exogenous netrin-1, neurons changed their vector angle within 4 h after treatment, a change that was significant whether alternative ectopic projections were included or excluded from the analysis (Fig. 11c). Neurons in anti-DCC- and in UNC5H2-Ig-treated tadpoles also remodeled and redirected their dendrites (Figs. 4f), effectively changing their vector and growth directionality within 4 h after treatment (Fig. 11c).

Fig. 10 Overlays of sample neurons at 0, 2, and 4 h illustrate changes in dendritic arbor morphology in response to treatment and between imaging intervals. a Confocal stacks of individual neurons from control, netrin-1-, UNC5H2-Ig-, and anti-DCC-treated tadpoles were reconstructed with MetaMorph creating three-dimensional wireframes of each stack. Wireframes were color-coded based on imaging time point (black, 0 h; blue, 2 h; red, 4 h), overlapped, and aligned over Scholl concentric circles with the primary dendrite placed at a 0° angle (X-axis; gray line). Dynamic changes in dendritic morphology every 2 h over a 4-h imaging period are illustrated by the emergence of blue (2 h) or red branches (4 h) from under the black wireframe (0 h). b, c Cumulative wireframes from a subset of seven neurons per condition better illustrate the dynamic changes in growth between the 0- and 2-h imaging interval (b), and the 0- and 4-h imaging interval (c), for each treatment group. Large arrows point to sample ectopic branches newly extended at the time point indicated by the color of the arrow (blue, 2 h; red, 4 h). Short arrows point to already established branches that changed their directionality of growth at the time point indicated by the color of the arrow (blue, 2 h; red, 4 h) Full size image

Fig. 11 Perturbations in tectal netrin levels or signaling alter dendritic arbor directionality. a Proportion of neurons that developed ectopic basal projections within the 24-h period in each group. b Angle analysis performed on tectal neuron arbors sums all branch points to produce a net vector. The angle change was calculated from the tangents of arbors from 0 to 4 h. c The change in dendritic arbor directionality is shown as the difference in angle for neurons from 0 to 4 h and was measured both including (with) and excluding (without) ectopic projections. d Proportion of stable branches with net angle change. The percentage of stable branches that individually changed their angle by at least 10° was calculated for a subset of randomly selected neurons (n = 4). The individual branch tip vectors for each branch were compared from 0 to 4 h to calculate the angle change. Note that a larger proportion of stable branches altered their angle in neurons following anti-DCC treatment when compared to all other groups. Statistical significance was by Kruskal-Wallis Friedman with Dunn’s multiple comparison test. Significance when compared to control is *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate SEM Full size image

The effects of netrin-1, UNC5H2-Ig, and anti-DCC treatments indicate that even though all of the manipulations in netrin signaling significantly impact growth directionality in a relatively similar way, the mechanisms responsible for this remodeling may differ. Specifically, neurons in tadpoles treated with netrin-1 or UNC5H2-Ig showed dynamic dendrite branch remodeling that differed from those in tadpoles treated with anti-DCC, since anti-DCC did not affect new branch addition or branch stabilization rates (Fig. 7). In vivo imaging showed that some neurons seemed to grow or reorient their branch(es) in a direction opposite to the neuropil in response to treatment (Fig. 12, see also Fig. 4, inserts). To further differentiate whether the change in directionality resulted primarily from a reorientation of stable branches or from the addition of new branches with a different angle of growth, we analyzed a subset of neurons that showed a significant change in net vector angle by at least 10°. For this analysis, we determined the vector angle of each individual branch tip for all branches at both 0 and 4 h to determine the proportion of stable branches that changed their vector angle by more than 10° for every neuron in each group. Significantly more of the stable branches changed their vector angles in neurons of netrin-1- or UNC5H2-Ig-treated tadpoles relative to controls (Fig. 11d, ANOVA, Dunnett’s multiple comparison test). Moreover, significantly more of the stable branches changed their vector angle in neurons in anti-DCC-treated tadpoles than in any other treatment group (anti-DCC vs. netrin-1 p < 0.05, and p < 0.01 vs. UNC5H2-Ig, ANOVA, Tukey’s multiple comparison test), indicating that manipulations in netrin signaling influence arbor directionality by reorienting stable dendrites, while branch retraction and new branch extension also contribute to the reorganization of the dendritic arbor when threshold netrin levels and/or signaling are changed.

Fig. 12 Individual branches change their orientation of growth in response to altered netrin levels. a, b The maximum projections of each confocal z-stack of two sample neurons at the 0-, 2-, and 4-h imaging time points, and the corresponding 90° view of each three-dimensional z-stack, illustrate the dynamic changes in growth and directionality of individual dendrites in response to acute netrin-1 treatment. The neuron in a corresponds to that shown in Fig. 5b. b’ For the sample neuron in b, a single primary dendrite and its individual secondary branches of the same branch can be discerned in the higher magnification images by selecting and projecting only the z-planes from each confocal stack that include that branch. By isolating the individual dendrite from the rest of the dendritic arbor, one can better differentiate the change in the direction of growth of the primary dendrite (short white arrows) that took place while some of its secondary branches were pruned (double blue arrows) or changed their direction of growth (green arrow) and others were maintained. Scale bars: 20 μm Full size image

We performed a number of correlational analyses to further determine a potential relationship between the degree of neuronal maturation and a neuron’s response to altered netrin levels or DCC signaling. No significant correlation between a number of morphological parameters measured prior to treatment (total branch number or length, DCI value) and type of response (increased branch addition, decreased branch stabilization, vector angle change, ectopic dendrite growth) was found for neurons in either netrin-1- or UNC5H2-Ig-treated tadpoles at 4 and 24 h. This suggests that actively branching tectal neurons respond to altered midbrain netrin levels independently of their maturational state. Only younger, newly differentiated neurons with total branch number and DCI below the average at the initial observation time point were more likely to grow an ectopic dendrite following anti-DCC treatment (branch number p = 0.0031, DCI value p = 0.0311; chi-square), suggesting that in addition to maintaining stable dendrites, DCC-mediated netrin signaling can prevent the formation of ectopic dendrites during early phases of dendritic growth when most remodeling occurs [20].

Sequestration of endogenous netrin-1 impacts visually guided behavior

Visual avoidance to moving light stimuli in Xenopus tadpoles is correlated with the maturation of visual responses in the optic tectum [21]. Deficits in visually guided behavior, in turn, have been correlated with abnormal visual system wiring [22, 23]. To test whether the netrin-induced changes in dendritic arbor morphology impact the functional organization of the retinotectal circuit, we used visually guided behavior as a functional assay. An avoidance behavior task [21] was adapted to probe specific visual responses of tadpoles at late stage 45 (Fig. 13a, see the “Methods” section). Tadpoles treated with netrin-1 or anti-DCC showed no changes in their ability to respond and avoid moving stimuli 4 h after treatment (Fig. 13b). In contrast to netrin-1 and anti-DCC, UNC5H2-Ig treatment resulted in abnormal visual avoidance behavior (Fig. 13b). Avoidance behavior of UNC5H2-Ig-treated tadpoles was significantly different from the behavior of the same tadpoles prior to treatment (0 h), as well as when compared to the behavior of tadpoles treated with either vehicle, netrin-1 or anti-DCC both at 0 and 4 h (avoidance at 0 h: vehicle 79.1 ± 3.2 %, UNC5H2-Ig 74.8 ± 4.5 %; avoidance at 4 h: vehicle 65.4 ± 2.91 %, UNC5H2-Ig 34.5 ± 7.2 %; p ≤ 0.005 two-way repeated measures ANOVA, n = 11–23 tadpoles per condition). The decreased ability of UNC5H2-Ig-treated tadpoles to respond to the moving stimuli was not due to alterations in their swimming capacity as the total swim time was not different for any of the treatment groups before or 4 h after treatment, whether tadpoles were presented with a moving dot (one-way ANOVA, not shown graphically, average swim time 57 ± 5.7 s out of 3-min total swim time/trial) or a video of a group of schooling tadpoles (data not shown). Consequently, sequestration of endogenous netrin-1 with UNC-5 ectodomain significantly influenced visually guided behavior in a rapid time scale, consistent with the significant dendrite remodeling effects and changes in tectal neuron morphology caused by the same treatment.