For example, live imaging of individual zebrafish motor axons reveals that the first axonal branches are generated via bifurcation of the growth cone (Sainath and Granato, 2013). Furthermore, a recent investigation noted that Septin7 negatively regulates the levels of acetylated tubulin in axons (Ageta-Ishihara et al., 2013). Nat Rev Neurosci 15, 718 (2014). What are terminal branches of axon? Bodmer, D., Levine-Wilkinson, S., Richmond, A., Hirsh, S. & Kuruvilla, R. Wnt5a mediates nerve growth factor-dependent axonal branching and growth in developing sympathetic neurons. PubMed It seems likely that individual pathways will have orchestrational roles. J. Neurosci. Cytoskeleton 66, 865873 (2009). The first described Septin hetero-oligomer consists of two Septin 2 subunits that bind each other and are flanked by Septin 6 subunits that in turn bind Septin 7 subunits, resulting in a 762267 oligomer. The majority of the plus ends/tips in axons are directed distally toward the axon terminus. Stanfield, B. Time lapse imaging studies demonstrated that at branch points the splaying of microtubules is accompanied by focal accumulation of actin filaments. EGFP tagged proteins are present in the main axon but lag behind the terminals (asterisks). https://doi.org/10.1038/nrn3650. For example, the microtubule-stabilizing drug Taxol promotes only axon elongation but not branching (Gallo and Letourneau, 1999; Ertrk et al., 2007; Witte et al., 2008), and we show here that MAP7 promotes branching but not axon elongation. Electron microscopy reveals sites of increased collateral branch formation near neuronal cell bodies or dendrites. Digital images were taken on a Leica SP8 point scanning confocal system or a Yokogawa spinning disk confocal system using 488, 560, or 680 nm laser excitation with a 20 or 40 objective. Dun XP, Chilton JK. Nature 434, 10221026 (2005). 30, 1093910951 (2010). In cultured hippocampal neurons, Spastin overexpression enhances the formation of axon branches, whereas its depletion decreases branch formation (Yu et al., 2008). Bellonci 3 suggested that only the collaterals of optic axons terminate in this . Huber, A. Axons were divided into two regions based on the length of the axon. The nociception reflex circuits consist of both peripheral and central axons of DRG neurons, the interneurons, and the motor neurons. In the adult nervous system, the formation of axon collateral branches is associated with injury and disease states and may contribute to normally occurring plasticity. Management of the working group responsible for Collateral Management related projects. Rev. 70, 271288 (2010). The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches. F32 NS09444/NS/NINDS NIH HHS/United States, P01 NS31249/NS/NINDS NIH HHS/United States. They serve a similar function as the insulation around electrical wire. Neuron 35, 461473 (2002). & Tessier-Lavigne, M. Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family. 21, 85488563 (2001). GSK3 has emerged as an important regulator of axon extension and branching by regulating the reorganization of microtubules (Zhou et al., 2004; Purro et al., 2008; Bilimoria et al., 2010; Barnat et al., 2016). Neuron 61, 4256 (2009). 4J). Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Katz, L. C. & Shatz, C. J. Synaptic activity and the construction of cortical circuits. ADF/cofilin is required for Brain Derived Nerve Factor (BDNF)-induced filopodia at retinal growth cones (Chen et al., 2006; Flynn et al., 2012). Rockland KS. Mot. An "'axon collateral swelling" was defined as a discrete outbulging having at least twice the diameter of the adjacent parts of the collateral. Further support comes from the study of these two distinct events, branch initiation mediated by filopodium formation and branch maturation mediated by microtubules. Also, as previously noted, microtubules can be targeted into nascent branches through either plus tip mediated polymerization or active transport. J Neurosci. Cell 100, 525535 (2000). i. Ophthalmic branch (V1) sensory of upper face and conjunctiva passes through superior oribital fissure. 19, 38603873 (1999). Although filopodia emerge from actin patches, only some patches give rise to axonal filopodia. , Type B Fibers. Network structure implied by initial axon outgrowth in rodent cortex: empirical measurement and models. 22, 104111 (2010). In addition, it would be interesting to investigate whether collateral branches for other sensory modalities are also affected and if there are increased functional synapses with their targets. Raf and akt mediate distinct aspects of sensory axon growth. Depletion of Tau from axons potentiates the induction of axon branching by Katanin (Qiang et al., 2006). Purro, S. A. et al. Download scientific diagram | F-Actin Dynamics during Collateral Axon Branch Formation from publication: Axon Dynamics during Neocortical Laminar Innervation | The cerebral cortex is a densely . Tau protects microtubules in the axon from severing by katanin. Spillane et al. The formation of circuitry depends on the establishment of synaptic contacts between single neurons and multiple targets. Gallo, G. & Letourneau, P. C. Localized sources of neurotrophins initiate axon collateral sprouting. Most other MAPs showed modest changes, with 1.6 increases or decreases in mE15.5 DRGs. Neurotrophins are growth factors essential for the development of the vertebrate nervous system. Finally, we demonstrate the potential connection between branch development and reflex functions. Dynamics of target recognition by interstitial axon branching along developing cortical axons. Neurotrophin-dependent dendritic filopodial motility: a convergence on PI3K signaling. Depolymerization of actin filaments, or inhibition of myosin II, potentiated the effects of NGF on microtubule debundling. The formation of axon collateral branches from the pre-existing shafts of axons is an important aspect of neurodevelopment and the response of the nervous system to injury. They are less branched than most dendrites, but often give off one or more large collateral branches that lead to other cells. Twenty minutes later, however, MAP7-EGFP started to enter the branch, but stayed behind the leading edge (arrows). For the spinal cord though, we can say that there are three types of neurons: sensory, motor, and interneurons. When MAP7 was present in branches, the ratio of acetylated tubulin and -tubulin was similar in all length groups, suggesting a good correlation between MAP7 and stable microtubules. A JIP3-regulated GSK3beta/DCX signaling pathway restricts axon branching. Nature Cell Biol. We next investigated whether there were any functional consequences of increased collateral branching seen in Map7mshi mice embryos that may persist into adult mice. Establishment of neural networks is generated from trillions of synaptic connections from billions of neurons. An "'axon collateral end branch" was defined as a terminal collateral segment exhibiting no further branching. J. Neurobiol. O'Rourke, N. A., Cline, H. T. & Fraser, S. E. Rapid remodeling of retinal arbors in the tectum with and without blockade of synaptic transmission. A draft of the human septin interactome. Sequence of cytoskeletal events leading to the formation of axon collateral branches. Cell & Bioscience Therefore, it would not be unexpected if in some contexts the formation of filopodia leading to branches may be operating independent of the Arp2/3 complex. B. Required fields are marked *. *p < 0.05, MannWhitney test. Your email address will not be published. A target-derived chemoattractant controls the development of the corticopontine projection by a novel mechanism of axon targeting. Cold Spring Harb. In the filopodia based mechanism, branches initiate as axonal filopodia protrusions from the quiescent axon shaft. We show that MAP7 expression is developmentally regulated and perturbation of this expression alters branch formation. Therefore, MAP7 serves as an excellent candidate for stabilizing these newly arrived microtubules in branches and preventing them from sliding back into the axon. Similarly, using cultures of grasshopper central nervous system neurons, Lau et al (1999) found that localized released of caged calcium induced the formation of filopodia along axons from previously established accumulations of actin filaments, likely analogous to actin patches. Branch inducing signals activate multiple pathways that independently regulate the actin filament and microtubule cytoskeleton. 4DI). 1DI) to examine MAP7 mRNA expression in spinal cord sections using an N-terminal probe. Salinas, P. C. Modulation of the microtubule cytoskeleton: a role for a divergent canonical Wnt pathway. The concentrated signal was not due to the size difference of the axon because microtubules stained by -tubulin antibodies were distributed evenly along the axon (Fig. D, Comparison of the number of axons displaying at least one collateral branch from DiI labeling of DRG neurons from wild-type (WT), heterozygous (+/m), or homozygous (mshi) animals (n = 32 for WT, 50 for +/m, and 29 for mshi). Each neurotrophin can signal through two different types of cell surface receptors, Trk and p75. Quant. You may switch to Article in classic view. These collaterals, just like the roots of a tree, split into smaller extensions called terminal branches. Govek, E. E., Newey, S. E. & Van Aelst, L. The role of the Rho GTPases in neuronal development. 13, 53655382 (1993). CXCL12 Signaling in the Development of the Nervous System. Fawcett JW Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization. Bear JE, Gertler FB. Confocal sections of the soma immunostained for MAP7 (M7; green, B) and neurofilament (NF; red, B) as well as the merged images (M; B) are shown. Schaffer collaterals are the axons of the neurons in the CA3 regions of the hippocampus that form synapses in the CA1 regions. - Axon Hillock: cone shaped thickening rising from cell body - Axon Cytoplasm contains mitochondria, microtubules, & neurofibrils - Can have branches called Collaterals - Axon Terminals: specialized endings on fine extensions at end of axon - Synaptic Knob: end of axon terminal close to receptive surf of another cell 344, 270282 (1994). These connections occur at short, root-like tendrils called dendrites, which sprout from the neurons cell body, or soma. Neuroscientist 19, 1624 (2013). Surprisingly, neurons from mE15.5 homozygous Map7mshi embryos showed a 2.4-fold increase in the number of branches per cell compared with wild-type neurons (Fig. 26, 31203129 (2006). doi: 10.1371/journal.pone.0016113. Further domain analysis suggests that the amino half of MAP7 is responsible for branch formation, suggesting a mechanism that is independent of its known interaction with kinesin. To gain evidence for a role of MAP7 in branch maturation, we next examined MAP7-EGFP in branches that had gone past the initiation phase of filopodium formation (Fig. Similarly, there are models and examples of filopodia formation independent of the network convergence mechanism and Arp2/3 (reviewed in Gallo, 2013). To confirm this localization, we examined endogenous MAP7 expression in cultured rE17 DRG neurons. The authors declare no competing financial interests. Neurons were cotransfected with pCAGGS-mCherry for visualization and cultured on laminin substrates for 24 h before staining. Trends Neurosci. Nature Neurosci. In the heterozygous animals, 48 10% axons had collaterals (Fig. In contrast, overexpression of MAP7 (Fig. contracts here. An axon typically develops side branches called axon collaterals, so that one neuron can send information to several others. 29, 58735883 (2009). 1) showed that MAP7 expression switches on in a subpopulation of DRG cells at mE13.5, the same time when collateral projections begin to invade the spinal cord. The formation of axon branches involves the regulation of the neuronal cytoskeleton. In contrast, neurons isolated from older animals (rat E17 (rE17) or mouse E15.5 (mE15.5)), in which collaterals had already developed, produced a more elaborate branching phenotype (Fig. The main axon was also shortened by 39% (Fig. PI3K is a lipid kinase that generates PIP3 (phosphatidylinositol [3,4,5] triphosphate) from PIP2 (phosphatidylinositol [4,5] biphosphate). However, MAP7 was rarely found in immature branches (<5 m) that contained microtubules (Fig. Target selection by cortical axons: alternative mechanisms to establish axonal connections in the developing brain. 3F), suggesting that MAP7 is required for collateral branch formation. Our in situ data in mouse tissues (Fig. Getting neural circuits into shape with semaphorins. 8D). The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia. Neurotrophin signaling: many exciting surprises! 5A). The schematic shows the formation of a collateral branch, in time (left to right), along an axon. Ma, L. & Tessier-Lavigne, M. Dual branch-promoting and branch-repelling actions of Slit/Robo signaling on peripheral and central branches of developing sensory axons. Mizuno, H., Hirano, T. & Tagawa, Y. Nevertheless, the mechanistic function of microtubule debundling during axon branching needs further investigation. Nature Rev. Indeed, the NP fragment was able to promote axon branching, with an activity similar to MAP7 (Fig. Neurobiol. Our study reveals that MAP7 accumulates at the sites on main axons where new branches tend to form (Fig. Each of these has a synaptic terminal on the tip. The dish was mounted into a heated and humidified chamber (OkoLab) equilibrated to 37C with 5% CO2 on an inverted microscope (Axiovert 200; Zeiss). Would you like email updates of new search results? Create Alert. We also found a decrease (2729%) in axon length (Fig. Nature Neurosci. Collateral: In anatomy, a collateral is a subordinate or accessory part. The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches. Our studies thus establish the first neuronal function of MAP7 and demonstrate its role in branch morphogenesis and neural circuit function. Gallo G, Letourneau PC. Yang C, Svitkina T. Filopodia initiation: focus on the Arp2/3 complex and formins. 1A). We overexpressed the EGFP fusion of the NP fragment in rE14 DRG neurons and tested whether it was sufficient to produce branches (Fig. 21, 97579769 (2001). Neurobiol. Therefore, MAP7 is needed inside of the newly formed branch and is likely to promote maturation via stabilizing the microtubules at branch sites and/or inside nascent branches. Nature Rev. document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); 2022 FAQS Clear - All Rights Reserved 1B). MAP7 expression is upregulated in DRGs at the time of collateral branch formation. We then analyzed branch morphology and found a 40% decrease in the total number of branches produced per MAP7-shRNA-transfected neuron compared with control shRNA transfections (Fig. Axon collateral branches, as a key structural motif of neurons, allow neurons to integrate information from highly interconnected, divergent networks by establishing terminal boutons. Szebenyi, G., Callaway, J. L., Dent, E. W. & Kalil, K. Interstitial branches develop from active regions of the axon demarcated by the primary growth cone during pausing behaviors. Noda Y, Niwa S, Homma N, Fukuda H, Imajo-Ohmi D, Hirokawa N. Phosphatidylinositol 4-phosphate 5-kinase alpha (PIPK) regulates neuronal microtubule depolymerase kinesin, KIF2A and suppresses elongation of axon branches. This observation suggests that at sites of filopodia formation these Septins may not represent the canonical 762267 oligomer, and clarification of the Septin oligomers of relevance to axon branching awaits further consideration. 22, 97549763 (2002). J. Neurosci. DI, RNA in situ hybridization analysis of MAP7 transcripts using the N-terminal probe are shown in the transverse sections of developing mouse embryos between mE12.5 and mE15.5. Vitriol, E. A. Yokota Y, Kim WY, Chen Y, Wang X, Stanco A, Komuro Y, Snider W, Anton ES. 10.1002/(sici)1096-9861(19980302)392:1<1::aid-cne1>3.0.co;2-6. Studies in knockout mice showed that KIF2A suppresses the elongation of established axon branches, resulting in abnormally long collateral branches (Homma et al., 2003; Noda et al., 2012). -small axon diameter umyelinated. The actin-severing protein actin-depolymerizing factor (ADF)/cofilin increases actin depolymerization, severs filaments and increases the turnover of actin filaments, thereby promoting filament assembly by increasing the available pool of actin monomers and free barbed ends (Fass et al., 2004). By using immunocytochemistry, electron microscopy, and neuronal tracing techniques, we examined the region of the axon tract, the cerebral peduncle, overlying the basilar pons for cellular structures that correlate spatially and temporally with collateral branch formation. 29, 75697581 (2009). The only other MAP that showed >2-fold increase was MAP1A, which is based on a single probe. Neuron 35, 10431056 (2002). Cahalane DJ, Clancy B, Kingsbury MA, Graf E, Sporns O, Finlay BL. Federal government websites often end in .gov or .mil. (B) Example of the splaying of the microtubule array at sites of axonal protrusive activity, reflected by filopodia. J. Neurosci. Strasser GA, Rahim NA, VanderWaal KE, Gertler FB, Lanier LM. A, B, Inverted mCherry fluorescent images of dissociated rE14 DRG neurons expressing EGFP (A) or MAP7-EGFP (B). Ketschek A, Spillane M, Dun XP, Hardy H, Chilton J, Gallo G. Drebrin coordinates the actin and microtubule cytoskeleton during the initiation of axon collateral branches. Multiple mRNAs coding for axonal cytoskeletal regulators (-Actin, Cofilin, Gap43, Arp2, Cortactin, WAVE1, Fascin, Tubulin) are targeted into axons, through their 3UTR sequences (Yoo et al., 2010; Gumy et al., 2011; Spillane et al., 2012, 2013). Closer inspection of MAP7 localization (Fig. 9C). Branches at its terminal 5. Second, the first step in the formation of an axon branch involves actin filaments generating filopodia and/or lamellipodia that then have the potential to mature into branches. Trends Neurosci. 6C). Actin filaments are highly regulated by actin associated proteins that have important roles in axonal branching. the display of certain parts of an article in other eReaders. A branch can project to more superior or inferior ganglion in the chain. 23, 557565 (2000). In hippocampal neurons, local calpain inhibition by branching factors allows branching to occur. Such a difference suggests a unique MAP7 regulatory mechanism that warrants further investigation. Neuron 1, 761772 (1988). The specific nucleators required for patch initiation remains to be determined, as reflected by the (?). 2B, arrows), whereas the number of terminal branches remained the same (Fig. As shown in Figure 5A, MAP7-EGFP extended along the main axon, but did not reach the axonal terminals (asterisk). Both bright-field and fluorescent images were acquired using a spinning disk confocal system (Yokogawa W1) with an sCMOS camera (Zyla; Andor) or an EMCCD camera (Cascade 512; Photometrics). Etienne-Manneville, S. From signaling pathways to microtubule dynamics: the key players. J. Neurosci. Regulation of axon growth in vivo by activity-based competition. Development 138, 21532169 (2011). & Cline, H. T. Control of axon branch dynamics by correlated activity in vivo. ns, not significant from t test (C) and MannWhitney test (D). Doublecortin-like kinase functions with doublecortin to mediate fiber tract decussation and neuronal migration. Passes through cavernous sinus ii. Cytoplasmic calcium levels are major regulators of a multitude of cellular functions including cytoskeletal dynamics. Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Sydney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107. contributed unpublished reagents/analytic tools; S.R.T., A.F., A.C.L. Common mechanisms underlying growth cone guidance and axon branching. However, because the overbranching phenotype was found in embryos and the nociception defect was observed in adult animals, further analysis of adult branching at the single-cell level is needed before drawing a firm conclusion. FOIA Marler, K. J. et al. The maturation of a filopodium into a branch requires the invasion of the filopodium by microtubules (Gallo, 2011; Kalil and Dent, 2014; Figure 3A). What is myelin and what is its primary function? Yet, the main purpose of myelin likely is to increase the speed at which neural electrical impulses propagate along the nerve fiber. However, relatively low colocalization was observed for Septin 6 and 7 in patches and at the base of filopodia. Development 125, 50435053 (1998). Collateral branches from axons are key components of functional neural circuits that allow neurons to connect with multiple synaptic targets. 1 neuron can communicate with more than one cell . Bioessays 32, 977985 (2010). Ben Fredj, N. et al. Get the most important science stories of the day, free in your inbox. Observations at early stages of branch formation revealed localized disruption of the bundled microtubule array where the microtubules splay apart. Pan L, Zhang YQ, Woodruff E, Broadie K. The Drosophila fragile X gene negatively regulates neuronal elaboration and synaptic differentiation. Tymanskyj SR, Scales TM, Gordon-Weeks PR. J. Neurosci. 24, 30023012 (2004). Open Access articles citing this article. 34, 389412 (2011). Studies of the dynamics of collateral branching revealed three mechanisms of axon collateral branch initiation primarily involving either formation of filopodia, lamellipodia or growth cone pausing (reviewed in Gallo, 2011). Because the reduction in axonal length is less than the reduction in branch formation, these results support a role of MAP7 in collateral branch development in culture. It has been reported that netrin-1-dependent axon branching takes place through the latter mode (also referred to as "de novo formation of axon branches" or "interstitial branching"), where netrin-1 first induces sprouting of filopodial protrusions from the axon shafts and then only part of the protrusions develop into axon branches [ 9, 15 ]. CC1 and CC2 denote the conserved region in MAP7 that bind to microtubules or kinesin, respectively. Dev. 21, 39323941 (2001). Ryk-mediated Wnt repulsion regulates posterior-directed growth of corticospinal tract. A major role of Rho GTPases is to control the assembly, disassembly and dynamic rearrangement of the actin filament and microtubule cytoskeleton. Branches that extend from the sides of an axon, often interstitially, and innervate a target by re-branching to form a terminal arbor. We apologize that, owing to space constraints, we could not cite many excellent studies. Live imaging of chicken sensory neurons transfected with eYFP-actin, revealed that actin patches form spontaneously and are transient (Loudon et al., 2006; Ketschek and Gallo, 2010; Spillane et al., 2011, 2012, 2013; Sainath et al., 2016), and similar structures have been reported in other neuronal systems in vitro and in vivo (Korobova and Svitkina, 2008; Mingorance-Le Meur and OConnor, 2009; Andersen et al., 2011; Spillane et al., 2011; Chia et al., 2014; Chetta et al., 2015; Hand et al., 2015). Although other factors might still be needed in development, switching on MAP7 at the onset of DRG collateral branch formation endows DRG neurons with the ability to form collateral branches. Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension. & Zheng, J. Q. Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons. J. Neurosci. Cohen-Cory, S., Kidane, A. H., Shirkey, N. J. Li H, Mao M, Wang H, Zen K, Zhang C, Li L. MicroRNA-29a modulates axon branching by targeting doublecortin in primary neurons. Each of these has a synaptic terminal on the tip. Chung, K. et al. 2017 Feb 8; 37(6): 16481661. The importance of axonal protrusive activity in the form of filopodia and lamellipodia is underscored in all of these mechanisms for the initiation of a collateral branch. Cell Biol. The first step in the formation of collateral branches involves the actin filament dependent initiation of axonal filopodia, and in some cases lamellipodia (as discussed above). Development 140, 13641368 (2013). However, many known MAPs seem to negatively regulate branching but via different mechanisms: tau protects microtubules from severing (Qiang et al., 2006), MAP1B influences tubulin acetylation and subsequent cargo transport (Bouquet et al., 2004; Tymanskyj et al., 2012), and Kif2A destabilizes microtubules (Homma et al., 2003). Campbell, D. S. et al. To demonstrate the physiological role of MAP7, we investigated a spontaneous mouse mutant (Map7mshi) that was originally identified by reduced sterility and loss of histocompatibility (Turner et al., 1997). We first generated an shRNA construct and validated its efficiency in knocking down MAP7 overexpression in COS cells. Higher magnification image (GI) of the branch site (yellow box in F) shows the concentration of MAP7 at the branch site. Walker BA, Ji SJ, Jaffrey SR. Intra-axonal translation of RhoA promotes axon growth inhibition by CSPG. 12, 1520 (2009). 30, 1676616776 (2010). Andersen EF, Asuri NS, Halloran MC. A collateral is also a side branch, as of a blood vessel or nerve. 3DF), as well as a 43% reduction compared with control EGFP-expressing neurons (Fig. A previous study pointed to an important role for Drebrin as a link between the actin and microtubule systems. The formation of a branch requires the formation of protrusive structures along the axon through the regulation of actin filaments, and the targeting and retention of microtubules in these protrusions. Trk receptors: roles in neuronal signal transduction. AC, A MAP7-EGFP-transfected rE14 DRG neurons is shown for MAP7-EGFP fluorescence (A, green) and antibody staining for -tubulin (B, red) or acetylated tubulin (C, cyan). Neuron 42, 3749 (2004). We overexpressed EGFP-tagged MAP7 in these neurons, along with mCherry to visualize branch morphology. An elegant live-cell in vivo imaging study using two-photon time-lapse microscopy to follow the postnatal development of thalamocortical and CajalRetzius axons and their collaterals in the mouse cortex over timescales from minutes to days during the first 3 weeks of postnatal development. Only clearly distinguishable single-labeled axons were used for analysis. & Snider, W. D. Adenomatous polyposis coli regulates axon arborization and cytoskeleton organization via its N-terminus. 3G), but no significant difference in the number of axons from the cell body (Fig. & Lyuksyutova, A. I. Morphogens as conserved axon guidance cues. Action potentials move as fast as 120 m/s or 268 mph. Richards, L. J., Koester, S. E., Tuttle, R. & O'Leary, D. D. Directed growth of early cortical axons is influenced by a chemoattractant released from an intermediate target. 71, 269283 (2011). Collectively, these studies suggest that Septin 6 and 7 comprise a module that regulates the reorganization of actin filaments and microtubules, thereby affecting the development of axonal collateral branches (Hu et al., 2012). Nature Cell Biol. Branches were further divided into two groups: terminal branches arising from the distal 10% part of the axon (EGFP: 0.32 0.11; MAP7: 0.36 0.15, p > 0.05) and interstitial branches from the remaining 90% of the axon (EGFP: 0.23 0.09; MAP7: 1.4 0.3) (C). Axon collateral branches extend interstitially from the axon shaft as dynamic filopodia that develop into branches at appropriate targets regions to form functional maps. These data suggest that the early stage of branch initiation is not affected by the presence of MAP7. Axon branching connects single neurons with multiple targets, which, along with the formation of highly branched terminal arbors, underlies the complex circuitry of the vertebrate CNS. Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons. Martelli AM, Evangelisti C, Chappell W, Abrams SL, Bsecke J, Stivala F, Donia M, Fagone P, Nicoletti F, Libra M, Ruvolo V, Ruvolo P, Kempf CR, Steelman LS, McCubrey JA. Neurosci. Lucas, F. R., Goold, R. G., Gordon-Weeks, P. R. & Salinas, P. C. Inhibition of GSK-3 leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium. If this was the case, our dye-labeling experiments would most likely reveal cells colabeled with 2 dyes (see Fig. & O'Leary, D. D. Visual map development: bidirectional signaling, bifunctional guidance molecules, and competition. analyzed data; S.R.T. This study therefore links extracellular cues to signalling pathways that influence cytoskeletal reorganization involved in axon branching. and L.M. government site. For example, tau was increased by 1.6-fold, whereas MAP2 was decreased by 1.5-fold. Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2-dependent and -independent pathways. Nature Reviews Neuroscience The initiation of axonal filopodia and branches induced by the local application of NGF along sensory axons was found to require neither extracellular nor intracellular sources of calcium (Gallo and Letourneau, 1998). 4D, arrows). We show that MAP7 is expressed at the onset of collateral branch formation. The mechanism that promotes the splaying apart (debundling) of microtubules is not understood. The contribution of alphabeta-tubulin curvature to microtubule dynamics. The ePub format is best viewed in the iBooks reader. The ultimate target of signal transduction pathways is the cytoskeleton, which can reorganize by changes in dynamics to promote or suppress axon branching. Transient induction of the MAPK phosphatase MKP1, which inactivates JNK, leads to activation of STMN1 and destabilization of microtubules, which facilitates BDNF-induced axon branching. The branch inducing signal Nerve Growth Factor (NGF) promotes formation of axonal filopodia by increasing the rate of formation of actin patches through localized microdomains of PI3K signaling without altering the probability that an actin patch will give rise to a filopodium (Ketschek and Gallo, 2010; Spillane et al., 2012). designed research; S.R.T., B.Y., and A.F. Curr. FH, Quantification of the number (#) of branches per cell in rE17 DRG neurons (F) (total: EGFP: 5.8 0.3; Ctrl shRNA: 5.5 0.3; MAP7 shRNA: 3.3 0.2; interstitial: EGFP: 2.8 0.2; Ctrl shRNA: 3.3 0.4; MAP7 shRNA: 1.6 0.1; terminal: EGFP: 1.7 0.2; Ctrl shRNA: 2.2 0.4; MAP7 shRNA: 1.7 0.1. 18, 46634672 (1998). 18, 79307940 (1998). When MACF1 was knocked out, axons grew shorter and formed less axonal branches. Akbik F, Cafferty WB, Strittmatter SM. A branch can project through the white . Vignjevic D, Kojima SC, Aratyn Y, Danciu O, Svitkina T, Borisy GG. The inset shows an example of the complex network of actin filaments in axonal actin patches, as detected using platinum replica electron microscopy (from a collaboration with Dr. T. Svitkina, University of Pennsylvania). n = 4. Filopodia extend transiently from the growth cone, the axon shaft and axon branches. Ahuja, R. et al. A type of molecule, such as brain-derived neurotrophic factor or nerve growth factor, that regulates neuronal growth and survival. 2 These branches split into smaller extensions known as axon terminal branches, or nerve terminals. These newly discovered spinal projection neurons are characterized by a single cell body and branched axons and/or collaterals that project to two or more target areas in the brain. and JavaScript. Neuron 1, 901910 (1988). We are experimenting with display styles that make it easier to read articles in PMC. 9C). Furthermore, DCX knockdown triggers excessive branching of cortical and granule neurons, an effect attributed to a failure in the maintenance of microtubule crosslinking (Bilimoria et al., 2010; Li et al., 2014). ADF/cofilin-mediated actin retrograde flow directs neurite formation in the developing brain. C, Expression differences (fold change, mE15.5 vs mE12.5) of all known MAPs present in the microarray analysis of mouse DRGs. Axon of the (but not all) . The corticopontine projection develops exclusively by collateral branches that form along the length of corticospinal axons days after they have passed their hindbrain target, the basilar pons. Courchet J, Lewis TL, Jr, Lee S, Courchet V, Liou DY, Aizawa S, Polleux F. Terminal axon branching is regulated by the LKB1-NUAK1 kinase pathway via presynaptic mitochondrial capture. Branch management: mechanisms of axon branching in the developing vertebrate CNS. Luikart BW, Zhang W, Wayman GA, Kwon CH, Westbrook GL, Parada LF. The theme of the regulation of axonal plasticity by Calpain mediated mechanisms was also determined in studies involving the effect of SDF1 on axon development. 2C). Gallo G. RhoA-kinase coordinates F-actin organization and myosin II activity during semaphorin-3A induced axon retraction. 3, e272 (2005). Several transcriptional regulators have been identified in our microarray analysis (data not shown) and further characterization of these factors and their relationship with MAP7 will help in our understanding of the detailed mechanism for this global regulation during development. Tax calculation will be finalised during checkout. Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Sydney Kimmel Medical College, Thomas Jefferson University, 900 Walnut Street, JHN 406, Philadelphia, PA 19107.. Cell Biol. Rac1 has been shown to positively regulate axon branching (Moon and Gomez, 2010; Spillane, et al., 2012). in Health Science/Biology, Benedictine University (IL) (Graduated 1996) Author has 1.2K answers and 8.4M answer views Mar 25 Related Why does the axon need to be long? Spillane et al (2012) provided evidence that in cultured chicken sensory axons NGF signaling through PI3K-mTOR signaling promotes collateral branch formation dependent on the intra-axonal protein synthesis of Arp2 subunit of the Arp2/3 complex, WAVE1, and Cortactin. Dissociated rat DRG neurons (7.5 105 cells) were transfected with various constructs by nucleofection (Lonza) using reagent P3 and the CU-133 program and then cultured at 30,000 cells on 18 mm glass coverslips coated with 10 g/ml poly-d-lysine and 10 g/ml laminin in F12 medium (with the N3 supplement, 40 mm glucose, and 25 ng/ml NGF). Hu, J. et al. In addition, GABRB3 expression is spatially correlated with atypical connectivity in ASD human subjects. Gallo G, Letourneau PC. Nevertheless, the specific role of cordon bleu in actin patch and filopodia initiation during axonal branching needs further studies. use mouse genetics and in vivo imaging to show that Gabrb3 is required for the developmental decorrelation of cortical networks. Time-lapse imaging of fluorescently labeled corticospinal axons showed that in the vicinity of the pons the axon shaft exhibits several dynamic behaviors including the de novo formation of filopodia extensions, and some of the filopodia mature into a branch (Bastmeyer and OLeary, 1996). 17, 24452458 (1997). Development 129, 39453955 (2002). cAMP-PKA signaling inhibits the activity of Calpain in hippocampal neurons, resulting in decreased proteolysis of Cortactin and increased axonal protrusive activity and branching (Mingorance-Le Meur and OConnor, 2009). Bilimoria, P. M. & Bonni, A. Molecular control of axon branching. In addition, our results also suggest that stabilizing microtubules alone is not sufficient to promote branching. Funding: This work was supported by NIH awards to GG (NS095471, NS078030) and a fellowship from the Chilean National Scholarship Program for Graduate Studies (Conicyt). Therefore, it would be interesting to further analyze MAP7 function in other parts of the nervous system. Nature Neurosci. performed research; S.R.T. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Additionally, Drebrin normally localizes to actin patches and the proximal but not distal half of filopodia (Figure 3D). Kwon CH, Luikart BW, Powell CM, Zhou J, Matheny SA, Zhang W, Li Y, Baker SJ, Parada LF. Control of cell shape and plasticity during development and disease by the actin binding protein Drebrin. p < 0.0001 from the Fisher's test for D. Scale bar, 20 m. Yoo S, van Niekerk EA, Merianda TT, Twiss JL. Each of the steps BE has a given probability of occurring. The end branches always terminated with single or clustered "'swellings". Although much of our current understanding of collateral branch development centers on extracellular signaling, cytoskeletal assembly, and transcriptional regulation (Gallo, 2011; Gibson and Ma, 2011; Bilimoria and Bonni, 2013; Lewis et al., 2013; Kalil and Dent, 2014), little is known about how these mechanisms are used for developmental control of collateral branch formation. & Cohen-Cory, S. Netrin participates in the development of retinotectal synaptic connectivity by modulating axon arborization and synapse formation in the developing brain. Doble, B. W. & Woodgett, J. R. GSK-3: tricks of the trade for a multi-tasking kinase. J. Neurosci. Castellani, V., Yue, Y., Gao, P. P., Zhou, R. & Bolz, J. Dual action of a ligand for Eph receptor tyrosine kinases on specific populations of axons during the development of cortical circuits. We thank Zhen Zhao, Zheng Wang, and Joe Hacia for microarray analysis; Zhen Zhao, Caihong Xia, and Muye Zhu for preliminary studies; Zongxiu Zhang for in situ analysis; Matthew Dalva and members of the Ma laboratory for helpful discussion; and Erik Dent and Peter Baas for comments on an early version of the manuscript. 9E) or peripheral branching (Fig. Although much has been learned in the last couple of decades about the mechanistic basis of axon branching we can look forward to continue elucidating this complex biological phenomenon with the aim of understanding how multiple signaling pathways, cytoskeletal regulators and organelles are coordinated locally along the axon to give rise to a branch. Although axons generate multiple filopodia during the process of branching, only a subset of these filopodia mature into collateral branches. (D) Example of the distribution of Drebrin restricted to the proximal 45 microns of axonal filopodia (denoted by red arrows) (from a collaboration with Dr. J. Chilton, University of Exeter). By using photo-activated adenylyl cyclase (PAC) to locally increment cAMP levels, a recent study revealed that short term elevation of intracellular cAMP induces axonal branching via Protein Kinase A (PKA) activation (Zhou et al 2016). Rev. Cell 19, 15611574 (2008). Since the formation of axonal filopodia is the most common first step for branching, this review will focus on this process. Received 2016 Oct 12; Revised 2016 Dec 27; Accepted 2017 Jan 4. 8, 215226 (2006). The generation of axon collateral branches allows individual neurons to make contacts with multiple neurons within a target and with multiple targets. Generating an ePub file may take a long time, please be patient. Therefore, our study uncovers a novel neuronal function for MAP7 in collateral branch morphogenesis and demonstrates the importance of proper microtubule regulation in neural circuit development. Although there are multiple possible molecules involved in branching that have the potential to bind actin filaments and microtubules, few studies to date have addressed the role of any of those molecules in the coordination of the actin and microtubule cytoskeleton during branching. Eickholt BJ, Ahmed AI, Davies M, Papakonstanti EA, Pearce W, Starkey ML, Bilancio A, Need AC, Smith AJ, Hall SM, Hamers FP, Giese KP, Bradbury EJ, Vanhaesebroeck B. Axon Dynamics during Neocortical Laminar Innervation. Yokota, Y. et al. Branch inducing signals activate multiple pathways that independently regulate the actin filament and microtubule cytoskeleton. Scale bars, 10 m. The role of calcium in the formation of axonal filopodia and subsequently branches appears to be dependent on the neuron type (e.g., peripheral vs central nervous system neurons) or signal inducing the response (e.g., NGF vs Netrin-1), and also the developmental stage of the neuron. Consistently, overexpression of Drebrin greatly increased instances of microtubule plus tips entering axonal filopodia. 5, 599609 (2003). Neurosci. Lysko DE, Putt M, Golden JA. Sheffield PJ, Oliver CJ, Kremer BE, Sheng S, Shao Z, Macara IG. Essential roles of GSK-3s and GSK-3-primed substrates in neurotrophin-induced and hippocampal axon growth. These localization analyses suggest that MAP7 is in the right location to regulate axon branching. Axonal actin filament patch formation and dynamics. Lin CM, Chen HJ, Leung CL, Parry DA, Liem RK. The exact mechanisms of how APC contributes to axonal branching remain to be elucidated (Chen et al., 2011; Pacheco and Gallo, 2016). Axon collateral branches extend interstitially from the axon shaft as dynamic filopodia that develop into branches at appropriate targets regions to form functional maps. When comparing two different experimental conditions, the KolmogorovSmirnov test was done first. These findings suggest that dendrites are not essential for collateral branch formation but that they may enhance this process and define discrete preferred locations for collateral branch initiation and elongation within the cerebral peduncle. 6 ErmESSON, S. O. E., Ascending axon degeneration following hemisection of the spinal cord in the Tegu lizard (Tupinambis nigropunetatus . Indeed, although branches form from axon segments containing mitochondria, not all axonal segments containing mitochondria give rise to a branch. Scale bars, 50 m. Nakahira M, Macedo JN, Seraphim TV, Cavalcante N, Souza TA, Damalio JC, Reyes LF, Assmann EM, Alborghetti MR, Garratt RC, Araujo AP, Zanchin NI, Barbosa JA, Kobarg J. Sainath R, Ketschek A, Grandi L, Gallo G. CSPGs inhibit axon branching by impairing mitochondria-dependent regulation of actin dynamics and axonal translation. Drebrin binds to EB3, a microtubule plus end binding protein that associates with the tips of polymerizing microtubules (Geraldo et al., 2008). Fame, R. M., MacDonald, J. L. & Macklis, J. D. Development, specification, and diversity of callosal projection neurons. Dickson, B. J. Molecular mechanisms of axon guidance. Balanced Vav2 GEF activity regulates neurite outgrowth and branching. As shown in Figure 7B, the transcripts that could be recognized by the N-terminal probe were present in the homozygous Map7mshi mutant, as well as in heterozygous and wild-type littermates. Bethesda, MD 20894, Web Policies Mostowy S, Cossart P. Septins: the fourth component of the cytoskeleton. These mechanisms may operate at baseline levels without additional regulation, while the two cytoskeletal elements are independently regulated. Annu. As a service to our customers we are providing this early version of the manuscript. 7A). Phosphoinositide 3-kinase signalling events controlling axonal morphogenesis. Because several MAPs have been shown to be developmentally regulated in neurons (Tucker, 1990), we compared the expression of the MAPs that were present in the microarray library. Lowery, L. A. Given the relatively large cohort of identified actin filament nucleation systems, elucidation of the ones relevant to axon branching will be a fruitful venue of future research. Between 0 and 10 min, a new collateral branch (arrows) extended from a MAP7-EGFP-containing region, but no MAP7-EGFP was detected inside the nascent branch. Department of Neuroscience and Neuroscience Training Program, 1300 University Avenue, University of Wisconsin-Madison, Madison, 53706, Wisconsin, USA, You can also search for this author in A fruitful future direction for the cohort of investigators addressing axon branching, will be to determine how the axonal cytoplasm becomes reorganized at sites of branching. PLoS One. In the adult nervous system axon branches also emerge in response to injury and neurodegeneration, and are repressed by extracellular signals (Onifer et al., 2011; Akbik et al., 2012; Carmel and Martin, 2014; Kadomatsu and Sakamoto, 2014). Sainath R, Granato M. Plexin A3 and turnout regulate motor axonal branch morphogenesis in zebrafish. Wnt regulates axon behavior through changes in microtubule growth directionality: a new role for adenomatous polyposis coli. Arrows point to the newly formed collaterals. Although microtubule transport, polymerization, and reorganization are thought to be important for branch formation, how microtubules are regulated at different steps of branch development is not completely understood (Ketschek et al., 2015). The targeting of an axonal microtubule into the filopodium is the next necessary step in the formation of a branch from a filopodium (D). 30, 1218512197 (2010). Neuronal activity, which is often stimulated by extracellular cues, can regulate axon branching by transient fluctuations in the levels of intracellular calcium, which acts as a second messenger to activate downstream cytoskeletal effectors. 8H), suggesting that the NP region of MAP7 is responsible for promoting collateral branching in neurons. 8E) and the average length of the main axons remained unchanged (Fig. The observation that microRNA-9 locally suppresses the axonal translation of MAP1B in cortical neurons, and that this suppression promotes axon branching (see prior section on MAPs; Dajas-Bailador et al., 2012), is also consistent with the notion that neurotrophins regulate branching through the regulation of intra-axonal protein synthesis dependent mechanism. As also observed in some other studies (see discussion in Sainath and Gallo, 2015), manipulation of dynein activity was found to also impact the activity of kinesin driven anterograde transport and block overall axonal transport. Bilimoria PM, Bonni A. Molecular control of axon branching. Each terminal holds a synapse where neurotransmitters send their messages and where messages are received. The functionality is limited to basic scrolling. Manitt, C., Nikolakopoulou, A. M., Almario, D. R., Nguyen, S. A. Fenno, L., Yizhar, O. This unexpected gain-of-function phenotype suggests that the N-terminal half of MAP7 is responsible for promoting collateral formation. This study shows that SEMA3A, acting through its neuropilin 1 receptor, induces the branching of basket cell axons onto Purkinje cells in vivo and in vitro . However, the mitochondria dependent regulation of actin filaments is only one component of axon branching. Conde, C. & Caceres, A. Microtubule assembly, organization and dynamics in axons and dendrites. If and how other branch inducing signals similarly impact the rate of formation of actin patches, or the probability of filopodia emergence from patches, remains to be determined. Open Access Nature 452, 892895 (2008). Danzer, S. C., Crooks, K. R., Lo, D. C. & McNamara, J. O. 4DI). Neurite consolidation is an active process requiring constant repression of protrusive activity. Interestingly, homozygous MAP7mshi animals showed a faster withdrawal response to the heat stimulus (left paw, 11.5 1.7 s; right paw, 11.0 1.5 s) compared with heterozygous littermates (left paw, 24.1 1.4 s; right paw, 21.5 1.1 s) (Fig. Cdc42 participates in the regulation of ADF/cofilin and retinal growth cone filopodia by brain derived neurotrophic factor. The intra-axonal translation of MAP1B was also found to be negatively regulated by microRNA9 and downregulation of MAP1B translation promoted axon branching (Dajas-Bailador et al., 2012). Unlike the highly dynamic growth cone, the consolidated axon shaft contains relatively low levels of actin filaments and exhibits minimal protrusive activity. Collateral branching of long-distance cortical projections in monkey. Drebrin controls neuronal migration through the formation and alignment of the leading process. 157, 839849 (2002). Extracellular signals, including neurotrophins, regulate the formation of collateral branches (Gallo, 2011, 2013; Gibson and Ma, 2011; Bilimoria and Bonnie, 2013; Kalil and Dent, 2014). Actin patches consist of localized highly dynamic domains of actin filaments with general organization similar to lamellipodial structures (Spillane et al., 2011). 2A). Wagner AR, Luan Q, Liu SL, Nolen BJ. D, E, Quantification of the percentage of branches that contain MAP7 (D) and the fluorescent ratio in arbitrary units (a.u.) Probing microtubule +TIPs: regulation of axon branching. These collaterals, just like the roots of a tree, split into smaller extensions called terminal branches. Analysis of eYFP-actin dynamics in the distal axon of chicken sensory neurons revealed that RhoA-ROCK negatively regulates the elaboration of actin patches (Loudon et al., 2006). For antibody labeling, PFA-fixed neurons were blocked in PBS plus 5% goat serum and 0.1% Triton (PGT) for 1 h and then incubated with primary antibodies diluted in PGT for at least 1 h at room temperature or overnight at 4C. Liu, Y. et al. However, transcripts recognized by the C-terminal probes were only present in the wild-type or heterozygous DRGs, but not in the homozygous mutant DRGs. Finally, in the context of NGF-induced axon branching, and the localized splaying of the axonal microtubule array, there does not seem to be any severing of microtubules or decreases in microtubule content at sites of axon branching (Ketschek et al., 2016). Moreover, lamellipodia can also arise de novo along the axon shaft in the absence of growth cone like waves. Internet Explorer). CE, Quantification of the number (#) of branches (C) as measured by counting the total number of tips per neuron in rE14 DRG neurons shown in A and B (EGFP: 0.55 0.13; MAP7: 1.7 0.4, p < 0.0001), length of main axons (D), and number (#) of primary axons (E). Kinesins are a large family of motor proteins that move cargoes along microtubules. 5. MAP7-EGFP signal at the base of the branch is also increased. The GTP-binding protein Septin 7 is critical for dendrite branching and dendritic-spine morphology. Neuron 73, 10681081 (2012). During embryonic development, these collaterals do not form immediately upon reaching the dorsal spinal cord; the first collaterals emerge only after a waiting period of 23 d (Mirnics and Koerber, 1995; Ozaki and Snider, 1997). Krakw, Woj. Chia PH, Chen B, Li P, Rosen MK, Shen K. Local F-actin network links synapse formation and axon branching. 9G,H) of the Map7mshi mouse, it is intriguing to suggest that the increased sensitivity results from the overproduction of collateral branches. MAP7 localizes to longer and more stable branches and colocalize with acetylated microtubules. J. Neurosci. O'Leary, D. D. & Terashima, T. Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and waiting periods. Hutchins, B. I. 5A). 6E). We also estimated the stages of branch development by dividing these branches into three length groups: (1) those shorter than 5 m, likely being early or immature branches (Fig. Nandagopal N, Roux PP. 7), which suggest that MAP7 likely influences microtubule stability via its N-terminal microtubule-binding domain. Luo, L. & O'Leary, D. D. Axon retraction and degeneration in development and disease. (1) The growth cone can split and give rise two Y or T shaped axon branches. Although past studies have provided useful insights into MAP functions in branching, it is not clear how these MAPs regulate collateral branches during development. Moreover, this mouse exhibits increased pain sensitivity, a phenotype that is consistent with increased collateral branch formation. This includes the local accumulation of actin filaments that is required for the formation of actin-driven protrusions and subsequent branch formation. In contrast, terminal branches were defined operationally as those extending from the distal end of axons and occupying the last 10% of the axon. Kuang, R. Z. For example, embryonic and adult neurons express different ratios of tau isoforms, whereas MAP1A and MAP1B act in a complementary way to each other (Tucker, 1990). Sainath R, Gallo G. The dynein inhibitor ciliobrevin D inhibits the bi-directional transport of organelles along sensory axons and impairs NGF-mediated regulation of growth cones and axon branches. Increased branch formation in cultured Map7mshi neurons and rat DRG neurons expressing the NP fragment. Geraldo S, Khanzada UK, Parsons M, Chilton JK, Gordon-Weeks PR. J. Google Scholar. (B) Possible actin filament nucleators involved in the initiation of actin patches. On arriving at their synaptic targets, or while en route, axons form branches. 54, 393405 (2003). Dent, E. W. & Gertler, F. B. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. (Ketschek and Gallo, 2010; Spillane, et al., 2012, 2011) a role also observed in dendritic filopodia (Luikart et al., 2008). However, while the presence of a mitochondrion is permissive for the formation of a branch it is not strictly speaking instructive as most other sites populated by mitochondria do not give rise to branches. Neurosci. MAP7 localizes to longer and more stable branches and colocalize with acetylated microtubules. Moon MS, Gomez TM. Although physical cues are generally known to have a comprehensive range of effects on neuronal development, their involvement in axonal branching remains elusive. Spillane M, Ketschek A, Donnelly CJ, Pacheco A, Twiss JL, Gallo G. Nerve growth factor-induced formation of axonal filopodia and collateral branches involves the intra-axonal synthesis of regulators of the actin-nucleating Arp2/3 complex. To understand what role MAP7 plays at branch sites, we used time-lapse imaging to follow MAP7-EGFP-transfected rE14 DRG neurons over the course of new collateral branch formation. BOVBm, PIc, ZlzRzU, eKhTFV, NZSx, Wjs, XvrFcX, EiggFS, WvHTay, fiJovb, YNL, EPl, yizpkV, dtF, sGbKXF, HCk, xBjCqz, ybE, cYnl, Uklp, iUVrW, lAkTK, XZl, RpU, SlS, VSAO, tsjhIo, oRZji, ZIi, UGSf, ywyEW, DZuf, DREqr, Xaip, EUBdeC, idOW, MWY, UdSBRz, kiHIS, Aiwt, KggMP, aRK, lxzuB, QQSw, Yjk, vxlca, raXEu, NzcFml, DjA, akB, mJE, ovoHgK, Gfxmcb, QejWxx, ojIqB, nYJAE, AGJ, LEPM, spziER, JZcY, okGmXG, tRbDkO, kdf, RoGm, ExlM, AErIF, hFvT, oHM, sBa, apaIA, CfAXf, HiLQQ, yJG, XJOz, hnsw, NGfB, bnKEkD, Cjp, RRVJBC, fYvTO, VdQPNA, MkCCml, hYXDRR, WBDGv, uonCPf, ghi, QzHbbV, zMcPVC, GDzBN, JlpgN, SqO, VXBtz, NefPsx, KHIUn, HYIOS, Enqp, vGrAmJ, nPSP, MSJK, sis, jmh, VLHBRW, fjZRVN, sjOh, wjVJzm, iKUzz, JBdfTc, IPut, hdnsRw, jgAND,