Bath-applied netrin-1 increases axon branching without affecting axon length, and locally applied netrin-1 induces filopodial protrusions on the axon shaft. A1, Examples of cortical neurons treated for 3 d with BSA (controls) and for 3 d with bath-applied netrin-1 (100 ng/ml) showing dramatic increase in axon branching with netrin-1. A2-A4, Bar graphs showing no increase in axon length but a significant increase in branch length (A2), a threefold increase in numbers of branches (A3), and a large increase in neurons with higher-order axon branches (A4) after netrin-1 treatment for 3 d. B, C, Sequences of time-lapse DIC images showing rapid development of axon branches after bath-applied netrin-1. B, C, An example of a branch extending from a growth cone and developing its own growth cone (B) and increased axon branching by filopodial protrusions (C). D-F, Locally applied netrin-1 induces rapid protrusion of filopodia (D, E) and lammelipodia (F) on the axon in the region of the pipette tip. G, Series of tracings of DIC images showing extension of transient filopodia during 15 min time period when netrin-1 is locally pulsed every 15 s (at top). Transient filopodia are illustrated as cumulative over time. When netrin-1 is withdrawn (bottom), filopodial protrusions cease. H, Graph plotting average growth of 12 axonal filopodia induced de novo from 10 neurons by netrin-1. Filopodia extend to 20 μm over 30 min.

Repetitive calcium transients in cortical axons are evoked by netrin-1 application within minutes. A, Netrin-1 evokes calcium activity in a previously silent axon. B, Netrin-1 increases the frequency and amplitude of calcium transients. C, BSA (control) elicits no calcium changes. Changes in fluorescence intensity are shown relative to baseline (F/F₀%), and measurements of calcium changes in all examples were obtained from axons and their growth cones. D, Graph comparing average frequency of calcium transients in 32 neurons before and after netrin-1 application. Frequency of calcium transients increases fourfold after netrin-1 application. E1-E5, DIC image (E1) and time-lapse images in pseudocolor (E2-E5), showing changes in calcium activity in a region of an axon after application of netrin-1. Calcium images were acquired every 5 s. E3, A calcium transient begins in the region of the arrowheads and is propagated along the axon in the direction indicated by the arrow. E2-E5, Regions of high calcium activity correspond to the region of the red and black arrowheads shown in the DIC image in E1. Measurements of frequencies and amplitudes of calcium transients in the two axon segments corresponding to the red (region I) and black (region II) arrowheads are shown in E6 and magnified in E7 for the time period of 6-8 min. Red arrows indicate time points when peak amplitudes occur only in axon region I. F1-F4, Localized pulsed application of netrin-1 can evoke calcium repetitive calcium transients in small localized regions of the axon. F4, Changes in frequency of calcium transients in the region of the axon bracketed in F3. G1-G4, DIC image (G1) and time-lapse images in pseudocolor (G2-G4) showing changes in calcium activity in an axon (green arrowhead) and its two branches (blue and red arrowheads) after local application of netrin-1 indicated by the green arrow. Localized calcium transients occur in one branch at 4 min (G3) and in the primary axon at 25 min (G4). Measurements of calcium transients in the axon (green) and in the branch (red) are shown during different time periods (G5-G7). Local transients in the branch are indicated by red arrows and are indicated in the primary axon by green arrow (see supplemental movie 1, available at www.jneurosci.org as supplemental material). As shown in G5-G7, calcium transients induced by netrin-1 can occur at different frequencies and asynchronously in an axon and its branches.

Calcium activity evoked by netrin-1 involves release from intracellular stores and is required for axon branching. A-C, Examples of neurons in the presence of netrin-1 treated with nifedipine to block L-type voltage-gated channels (A), 2-aminoethoxydiphenyl borate (2-APB) to block IP₃ receptors (B), and dantrolene to block ryanodine receptors (C). Neurons treated with 2-APB and dantrolene, but not nifedipine, show attenuation of calcium activity over time. D, Examples of neurons cultured with and without calcium in the medium in the presence of 0.5% BSA (control) or 100 ng/ml netrin-1. In the absence of extracellular calcium, netrin-1 does not promote axon branching, but axons are slightly longer. E, Examples of neurons treated with netrin-1 or with netrin-1 and the addition of nifedipine, 2-APB, or dantrolene, demonstrating that blocking calcium release from intracellular stores diminishes axon branching. F-H, Bar graphs comparing axon length, branch length, and numbers of branches per neuron in all experimental conditions shown in the examples in D and E.

Localized netrin-1 application evokes repetitive calcium transients that are simultaneous with development of axon branches in the same region of the axon. A, DIC time-lapse images showing protrusion of filopodia in response to local application of netrin-1. Arrows indicate three different branches. The branch at the arrow begins as a short filopodium but by 20 min has elongated and developed a complex growth cone. B, The corresponding time-lapse fluorescence images show high levels of repetitive calcium transients (see supplemental movie, available at www.jneurosci.org as supplemental material) that coincide with newly developing branches. As can be seen in the movie, calcium activity begins before filopodial protrusion and continues during development of branches over 20 min. C, Measurements of calcium transients in the primary axon and the three branches corresponding to lines of the arrows. The growth cone developing from the region of arrow a shows the largest changes in calcium activity.

Netrin-1 activates both CaMKII and MAPK. A, Examples of neurons treated with 0.5% BSA (control) for 15 min, KCl for 5 min, or netrin-1 for 15, 30, or 60 min and then immunostained with an antibody to CaMKII phosphorylated at the site of threonine 286. B, Bar graphs comparing average fluorescence intensity of immunostained neurons subjected to treatments described in A. C, Examples of neurons (shown in DIC) imaged first for 15 min to measure calcium activity after application of netrin-1, followed by rapid fixation and immunostaining of phospho-CaMKII. Intense staining only occurred in neurons showing repetitive netrin-1-induced calcium transients (red arrowhead points to growth cone of neuron with high calcium activity) but not in neurons that failed to show calcium activity in response to netrin-1 (blue arrow). Measurements of calcium activity were obtained from neurons indicated by red and blue arrowheads. D, Examples of neurons treated with 0.5% BSA (control) for 15 min, KCl for 5 min, netrin-1 for 15 min with or without the MAPK inhibitor U0126, or netrin-1 for 30 or 60 min followed by immunostaining with an antibody to the phosphorylated MAPK, MEK1/2. E, Bar graphs comparing average fluorescence intensity of immunostained neurons subjected to treatments described in B. F, Western blot showing increases in phosphorylated CaMKII after application of netrin-1 for 15 min or KCl for 5 min. Syntaxin was used to control for loading of the same amount of protein sample.

Pharmacological inhibition of CaMKII reduces axon outgrowth and branching. A, Examples of neurons treated with BSA (control) or netrin-1 with or without KN62, a general inhibitor to CaM kinases. B, C, Bar graphs comparing axon length and branch length (B) and number of branches per neuron (C) in all conditions. KN62 reduces both spontaneous and netrin-1-induced branching as well as axon length. D, Examples of neurons treated with netrin-1 with or without AIPII, a specific inhibitor to CaMKII. E-G, Bar graphs comparing axon length and branch length (E), number of branches (F), and percentages of neurons with first-, second-, and third-order branches in all conditions. AIPII in the presence of netrin-1 reduces axon and branch length as well as primary and higher-order branching.

Overexpression of αCaMKII promotes axon branching and increases axon length. A, B, Examples of neurons transfected with EGFP-tubulin (A) and EGFP-αCaMKII (B). Neurons overexpressing αCaMKII develop highly branched arbors similar to those after netrin-1 treatment. C, D, Bar graphs comparing axon length and branch length (C) and number of branches per neuron (D) in neurons overexpressing tubulin, αCaMKII, βCAMKII, and two mutant forms of αCaMKII (T286A and T286D) mutated at the threonine 286 autophosphorylation site. Overexpression of wild-type αCaMKII alone promotes axon branching. E-G, Examples of neurons transfected with EGFP alone (E), EGFP and a constitutively active (CA) mutant αCaMKII (F), and EGFP-CaMKIIKIIN, a CaMKII inhibitory protein (G). H, I, Bar graphs comparing axon length and branch length (H) and number of branches per neuron (I) in all conditions. J-N, Examples of neurons transfected with EGFP-tubulin (J), EGFP-αCaMKII (K), and EGFP-αCaMKII with addition of KN 62 (L), U0126 (M), or netrin-1 (N). O, P, Bar graphs comparing axon length and branch length (O) and number of branches per neuron in all conditions. KN62 completely abolishes the branch-promoting effects of αCaMKII in contrast to partial reduction with U0126, and netrin-1 slightly enhances the branching effects of αCaMKII.

Molecular and RNAi knockdown of CAMKII abolishes netrin-1-induced axon branching. A-C, Examples of neurons transfected EGFP (A), EGFP and treated with netrin-1 (B), and EGFP-CaMKIIN and treated with netrin-1. D, E, Bar graphs comparing axon length and branch length (D) and number of branches per neuron (E) in all conditions. Netrin-1 cannot promote branching in neurons overexpressing the inhibitory CaMKIIN protein. F1-F3, Example of a neuron (arrowheads) at 1 d in culture cotransfected with EGFP and a control DNA vector U6 (F1) and immunostained with an antibody to αCaMKII at 3 d in culture (F2). This same neuron, when stimulated with netrin-1, is able to branch extensively (F3). Thus, the control U6 vector does not affect levels of CaMKII or the ability of the axon to branch in the presence of netrin-1. G1-G3, Example of a neuron (arrowheads) at 1 d in culture cotransfected with EGFP and a U6-αCaMKII RNAi vector (G1). The same neuron is shown in a different fluorescent channel (G2) demonstrating immunostaining with an antibody to αCaMKII at 3 d in culture. The neuron (arrowhead) after RNAi knockdown of CaMKII has reduced levels of αCaMKII compared with the neighboring cells and concomitantly few axon branches (G3) when stimulated with netrin-1. H, Bar graph comparing fluorescence intensity of αCaMKII immunostaining in neurons transfected with the control U6 vector or the RNAi U6-αCaMKII. I, J, Bar graphs comparing axon length and branch length (I) and number of branches per neuron (J) in all conditions.

MAPK overexpression promotes and pharmacological or molecular inhibition of MAPK prevents netrin-1-induced axon branching without affecting axon outgrowth. A, Examples of neurons treated with BSA (control), netrin, and the MAPK inhibitors U0126 and PD98059 in combination with netrin and the CAMKII inhibitor KN62. B, C, Bar graphs comparing axon length and branch length (B) and number of branches per neuron (C) in all conditions. Both MAPK inhibitors prevent netrin-1-induced branching, and the combination of UO126 with KN62 severely reduces axon outgrowth and abolishes netrin-1-induced branching. D-I, Examples of neurons transfected with EGFP alone (D), EGFP and a constitutively active form of MEK1 (E), myc-tagged Erk2-MEK1-LA (F), a control myc-tagged Erk2-MEK1 (G), EGFP with the addition of netrin-1 (H), and EGFP with a dominant-negative form of Ras (RasN17) in the presence of netrin-1 (I). J-M, Bar graphs comparing axon length and branch length (J, L) and number of branches per neuron (K, M) in all conditions.