Visual experience and age affect synaptic organization in the mushroom bodies of the desert ant Cataglyphis fortis

Desert ants of the genus Cataglyphis undergo an age‐related polyethism from interior workers involved in brood care and food processing to short‐lived outdoor foragers with remarkable visual navigation capabilities. The quick transition from dark to light suggests that visual centers in the ant's brain express a high degree of plasticity. To investigate structural synaptic plasticity in the mushroom bodies (MBs)—sensory integration centers supposed to be involved in learning and memory—we immunolabeled and quantified pre‐ and postsynaptic profiles of synaptic complexes (microglomeruli, MG) in the visual (collar) and olfactory (lip) input regions of the MB calyx. The results show that a volume increase of the MB calyx during behavioral transition is associated with a decrease in MG numbers in the collar and, less pronounced, in the lip. Analysis of tubulin‐positive profiles indicates that presynaptic pruning of projection neurons and dendritic expansion in intrinsic Kenyon cells are involved. Light‐exposure of dark‐reared ants of different age classes revealed similar effects. The results indicate that this structural synaptic plasticity in the MB calyx is primarily driven by visual experience rather than by an internal program. This is supported by the fact that dark‐reared ants age‐matched to foragers had MG numbers comparable to those of interior workers. Ants aged artificially for up to 1 year expressed a similar plasticity. These results suggest that the high degree of neuronal plasticity in visual input regions of the MB calyx may be an important factor related to behavior transitions associated with division of labor. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 408–423, 2010     

Worker brain development and colony organization in ants: Does division of labor influence neuroplasticity?

Brain compartment size allometries may adaptively reflect cognitive needs associated with behavioral development and ecology. Ants provide an informative system to study the relationship of neural architecture and development because worker tasks and sensory inputs may change with age. Additionally, tasks may be divided among morphologically and behaviorally differentiated worker groups (subcastes), reducing repertoire size through specialization and aligning brain structure with task‐specific cognitive requirements. We hypothesized that division of labor may decrease developmental neuroplasticity in workers due to the apparently limited behavioral flexibility associated with task specialization. To test this hypothesis, we compared macroscopic and cellular neuroanatomy in two ant sister clades with striking contrasts in worker morphological differentiation and colony‐level social organization: Oecophylla smaragdina , a socially complex species with large colonies and behaviorally distinct dimorphic workers, and Formica subsericea , a socially basic species with small colonies containing monomorphic workers. We quantified volumes of functionally distinct brain compartments in newly eclosed and mature workers and measured the effects of visual experience on synaptic complex (microglomeruli) organization in the mushroom bodies—regions of higher‐order sensory integration—to determine the extent of experience‐dependent neuroplasticity. We demonstrate that, contrary to our hypothesis, O. smaragdina workers have significant age‐related volume increases and synaptic reorganization in the mushroom bodies, whereas F. subsericea workers have reduced age‐related neuroplasticity. We also found no visual experience‐dependent synaptic reorganization in either species. Our findings thus suggest that changes in the mushroom body with age are associated with division of labor, and therefore social complexity, in ants. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1072–1085, 2017     

Does behavioral flexibility compensate or constrain colony productivity? Relationship among age structure,labor allocation,and production of workers in ant colonies

Temporal change in colonial labor allocation, age structure, eclosion of workers, and numbers of eggs, larvae, and pupae in colonies of the ponerine ant,Diacamma sp., from Japan was surveyed for more than a year. Fluctuation of brood indicated that temporal variation in eclosion is caused by brood raising, not by cessation of egg supply. Labor allocation patterns at a given moment were related to subsequent colonial productivity by multiple linear regression. Fluctuating eclosion patterns can be predicted from labor allocation patterns extrapolated from their age structure, without considering behavioral flexibility. The predictions agree with observed patterns. This suggests that behavioral flexibility does not fully compensate for the deterioration in productivity caused by the fluctuation in age structure.     

Astrocytes Play a Key Role in Drosophila Mushroom Body Axon Pruning

Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-β ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. Here we show that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, we show that astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, we found that blocking the expression of the cell engulfment receptor Draper in astrocytes only affects axonal debris clearance. Thereby we uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. Our study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning.     

Transplantation of photoreceptor precursor cells into the retina of an adult Drosophila

Blindness caused by the disconnection between photoreceptor cells and the brain can be cured by restoring this connection through the transplantation of retinal precursor neurons. However, even after transplanting these cells, it is still unclear how to guide the axons over the long distance from the retina to the brain. To establish a method of guiding the axons of transplanted neurons, we used the Drosophila visual system. By testing different conditions, including the dissociation and preincubation length, we have successfully established a method to transplant photoreceptor precursor cells isolated from the developing eye discs of third‐instar larvae into the adult retina. Moreover, we overexpressed N‐cadherin (CadN) in the transplant, since it is known to be broadly expressed in the optic lobe well after developmental stages, continuing through adult stages. We found that promoting the cell adhesive properties using CadN enhances the axonal length of the grafted photoreceptor neurons and therefore is useful for future transplantation. We tested the overexpression of a CadN::Frazzled chimeric receptor and found that there was no difference in axonal length from our wild‐type transplants, suggesting that the intracellular domain of CadN is necessary for axonal elongation. Altogether, using the Drosophila visual system, we have established an excellent platform for exploring the molecules required for proper axon extension of transplanted neuronal cells. Future studies building from this platform will be useful for regenerative therapy of the human nervous system based on transplantation.     

Neuronal Plasticity in the Mushroom‐Body Calyx of Bumble Bee Workers During Early Adult Development

Division of labor among workers is a key feature of social insects and frequently characterized by an age‐related transition between tasks, which is accompanied by considerable structural changes in higher brain centers. Bumble bees (Bombus terrestris), in contrast, exhibit a size‐related rather than an age‐related task allocation, and thus workers may already start foraging at two days of age. We ask how this early behavioral maturation and distinct size variation are represented at the neuronal level and focused our analysis on the mushroom bodies (MBs), brain centers associated with sensory integration, learning and memory. To test for structural neuronal changes related to age, light exposure, and body size, whole‐mount brains of age‐marked workers were dissected for synapsin immunolabeling. MB calyx volumes, densities, and absolute numbers of olfactory and visual projection neuron (PN) boutons were determined by confocal laser scanning microscopy and three‐dimensional image analyses. Dark‐reared bumble bee workers showed an early age‐related volume increase in olfactory and visual calyx subcompartments together with a decrease in PN‐bouton density during the first three days of adult life. A 12:12  h light‐dark cycle did not affect structural organization of the MB calyces compared to dark‐reared individuals. MB calyx volumes and bouton numbers positively correlated with body size, whereas bouton density was lower in larger workers. We conclude that, in comparison to the closely related honey bees, neuronal maturation in bumble bees is completed at a much earlier stage, suggesting a strong correlation between neuronal maturation time and lifestyle in both species.     

Autumnal moth – why autumnal?

1. As for some other spring‐feeding moths, adult flight of Epirrita autumnata (Lepidoptera: Geometridae) occurs in late autumn. Late‐season flight is a result of a prolonged pupal period. Potential evolutionary explanations for this phenological pattern are evaluated. 2. In a laboratory rearing, there was a weak correlation between pupation date and the time of adult emergence. A substantial genetic difference in pupal period was found between two geographic populations. Adaptive evolution of eclosion time can thus be expected. 3. Metabolic costs of a prolonged pupal period were found to be moderate but still of some ecological significance. Pupal mortality is likely to form the main cost of the prolonged pupal period. 4. Mortality rates of adults, exposed in the field, showed a declining temporal trend from late summer to normal eclosion time in autumn. Lower predation pressure on adults may constitute the decisive selective advantage of late‐season flight. It is suggested that ants, not birds, were the main predators responsible for the temporal trend. 5. Egg mortality was estimated to be low; it is thus unlikely that the late adult period is selected for to reduce the time during which eggs are exposed to predators. 6. In a laboratory experiment, oviposition success was maximal at the time of actual flight peak of E. autumnata, however penalties resulting from sub‐optimal timing of oviposition remained limited.     

Division of labor in the hyperdiverse ant genus Pheidole is associated with distinct subcaste- and age-related patterns of worker brain organization

The evolutionary success of ants and other social insects is considered to be intrinsically linked to division of labor among workers. The role of the brains of individual ants in generating division of labor, however, is poorly understood, as is the degree to which interspecific variation in worker social phenotypes is underscored by functional neurobiological differentiation. Here we demonstrate that dimorphic minor and major workers of different ages from three ecotypical species of the hyperdiverse ant genus Pheidole have distinct patterns of neuropil size variation. Brain subregions involved in sensory input (optic and antennal lobes), sensory integration, learning and memory (mushroom bodies), and motor functions (central body and subesophageal ganglion) vary significantly in relative size, reflecting differential investment in neuropils that likely regulate subcaste- and age-correlated task performance. Worker groups differ in brain size and display patterns of altered isometric and allometric subregion scaling that affect brain architecture independently of brain size variation. In particular, mushroom body size was positively correlated with task plasticity in the context of both age- and subcaste-related polyethism, providing strong, novel support that greater investment in this neuropil increases behavioral flexibility. Our findings reveal striking levels of developmental plasticity and evolutionary flexibility in Pheidole worker neuroanatomy, supporting the hypothesis that mosaic alterations of brain composition contribute to adaptive colony structure and interspecific variation in social organization.     

Early Exposure to Ketamine Impairs Axonal Pruning in Developing Mouse Hippocampus

Mounting evidence suggests that prolonged exposure to general anesthesia (GA) during brain synaptogenesis damages the immature neurons and results in long-term neurocognitive impairments. Importantly, synaptogenesis relies on timely axon pruning to select axons that participate in active neural circuit formation. This process is in part dependent on proper homeostasis of neurotrophic factors, in particular brain-derived neurotrophic factor (BDNF). We set out to examine how GA may modulate axon maintenance and pruning and focused on the role of BDNF. We exposed post-natal day (PND)7 mice to ketamine using a well-established dosing regimen known to induce significant developmental neurotoxicity. We performed morphometric analyses of the infrapyramidal bundle (IPB) since IPB is known to undergo intense developmental modeling and as such is commonly used as a well-established model of in vivo pruning in rodents. When IPB remodeling was followed from PND10 until PND65, we noted a delay in axonal pruning in ketamine-treated animals when compared to controls; this impairment coincided with ketamine-induced downregulation in BDNF protein expression and maturation suggesting two conclusions: a surge in BDNF protein expression “signals” intense IPB pruning in control animals and ketamine-induced downregulation of BDNF synthesis and maturation could contribute to impaired IPB pruning. We conclude that the combined effects on BDNF homeostasis and impaired axon pruning may in part explain ketamine-induced impairment of neuronal circuitry formation.     

Plant killing by Neotropical acacia ants: ecology,decision‐making,and head morphology

Mutualistic species often associate with several partners that vary in the benefits provided. In some protective ant–plant mutualisms, ants vary in the extent at which they kill neighboring vegetation. Particularly, in acacia ants (Pseudomyrmex), the area around the host tree that ants keep free from vegetation (“clearings”) vary depending on the species. This study assessed whether interspecific variation in clearing size corresponds to workers biting on plant tissue of different thickness. As expected, workers from species making the largest clearings bit more often on thicker plant tissues than workers from species making smaller clearings. Because head shape affects mandible force, I also assessed whether pruning on thick tissue in mutualistic ant species or being a predator in non‐mutualistic species correlated with broader heads, which yield stronger mandible force. The species with the broader heads were non‐mutualistic predators or mutualistic pruners of thick tissues, which suggest that pruning neighboring vegetation in non‐predatory species demands force even when the ants do not kill prey with their mandibles. The findings reveal that clearing size variation in mutualistic ant partners of plants can also be observed at the level of individual decision‐making processes among workers, and suggest that head morphology could be a trait under selection in protective ant–plant mutualisms. Abstract in Spanish is available with online material.     

Age‐dependent and task‐related volume changes in the mushroom bodies of visually guided desert ants,Cataglyphis bicolor

Desert ants of the genus Cataglyphis are skillful long‐distance navigators employing a variety of visual navigational tools such as skylight compasses and landmark guidance mechanisms. However, the time during which this navigational toolkit comes into play is extremely short, as the average lifetime of a Cataglyphis forager lasts for only about 6 days. Here we show, by using immunohistochemistry, confocal microscopy, and three‐dimensional reconstruction software, that even during this short period of adult life, Cataglyphis exhibits a remarkable increase in the size of its mushroom bodies, especially of the visual input region, the collar, if compared to age‐matched dark‐reared animals. This task‐related increase rides on a much smaller age‐dependent increase of the size of the mushroom bodies. Due to the variation in body size exhibited by Cataglyphis workers we use allometric analyses throughout and show that small animals exhibit considerably larger task‐related increases in the sizes of their mushroom bodies than larger animals do. It is as if there were an upper limit of mushroom body size required for accomplishing the ant's navigational tasks. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Age‐related and light‐induced plasticity in opsin gene expression and in primary and secondary visual centers of the nectar‐feeding ant Camponotus rufipes

Camponotus rufipes workers are characterized by an age‐related polyethism. In the initial weeks of adult life, young workers perform tasks inside the nest before they switch to multimodal foraging tasks outside. We tested the hypothesis that this transition is accompanied by profound adaptations in the peripheral and central visual systems. Our results show that C. rufipes workers of all tested ages (between 1 and 42 days) express three genes encoding for ultraviolet (UV), blue (BL), and long‐wavelength (LW1) sensitive opsins in their retina, which are likely to provide the substrate for trichromatic color vision. Expression levels of all three opsin genes increased significantly within the first two weeks of adulthood and following light exposure. Interestingly, the volumes of all three optic neuropils (lamina, medulla, and lobula) showed corresponding volume increases. Tracing of connections to higher visual centers in the mushroom bodies (MBs) revealed only one optic pathway, the anterior superior optic tract, emerging from the medulla and sending segregated input to the MB‐calyx collar. The MB collar volumes and densities of synaptic complexes (microglomeruli, MGs) increased with age. Exposure to light for 4 days induced a decrease in MG densities followed by an increase after extended light exposure. This shows that plasticity in retinal opsin gene expression and structural neuroplasticity in primary and secondary visual centers comprise both “experience‐independent” and “experience‐dependent” elements. We conclude that both sources of plasticity in the visual system represent important components promoting optimal timing of the interior–forager transition and flexibility of age‐related division of labor. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1041–1057, 2016     

Effects of photoperiod and temperature on the rhythm and free‐running of adult eclosion in the Indian meal moth Plodia interpunctella

The rhythm of adult eclosion in the Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) is investigated under various photoperiods and temperatures aiming to determine the nature of the temperature compensation and the free‐running period. Insects that are committed to a nondiapause larval development show diel rhythms of adult eclosion at 30, 25 and 20 °C. At 30 °C, the eclosion peak (i.e. the mean time of eclosion) occurs approximately 20 h after lights off under an LD 4 : 20 h photocycle, and at approximately 15 h under an LD 20 : 4 h photocycle. At 25 °C, the peak of eclosion occurs approximately 19 h after lights off under an LD 2 : 20 h photocycle and at approximately 16 h under an LD 20 : 4 h photocycle. At 20 °C, the eclosion peak is significantly advanced under long days of >12 h (i.e. approximately 20 h after lights off under an LD 4 : 20 h photocycle and approximately 9 h under an LD 20 : 4 h photocycle), indicating an effect of both lights‐off and lights‐on signals on the timing of the adult eclosion. To determine the involvement of a self‐sustained oscillator, the rhythm of adult eclosion is examined under darkness at different temperatures (30 to 21 °C). The mean free‐running periods are 22.4, 22.8, 22.0 and 22.5 h at 30, 24, 23 and 22 °C, respectively, indicating that the eclosion rhythm is temperature‐compensated. However, this rhythm does not free‐run under constant darkness at 21 °C. Because a clear diel rhythm is observed under 24‐h photocycles at 20 °C, the oscillator might be damped out within 24 h at the lower temperature.     

Plant Ontogeny as a Conditionality Factor in the Protective Effect of Ants on a Neotropical Tree

The outcome of any interspecific interaction is often determined by the ecological context in which the interacting species are embedded. Plant ontogeny may represent an important source of variation in the outcome of ant–plant mutualisms, as the level of investment in ant rewards, in alternative (non‐biotic) defenses, or both, may be modulated by the plant's developmental stage. In addition, the abundance and identities of the ants involved in the interaction may change during ontogeny of the host‐plant. Here, we evaluated if plant ontogeny affects the interaction between ants and a savanna tree species (Caryocar brasiliense) that produces extrafloral nectar. We found fewer ants per branch and fewer species of ants per tree in juvenile than in reproductive trees of medium and large size. In addition, large‐sized reproductive trees were more likely to host more aggressive ants than were medium‐sized reproductive or juvenile trees. Such differences strongly affected the outcome of the interaction between ants and their host‐plants, as the magnitude of the effect of ants on herbivory was much stronger for large trees than for juvenile ones. The fact that we did not find significant ontogenetic variation in the concentration of leaf tannins suggests that the observed differences in herbivory did not result from a differential investment in chemical defenses among different‐sized plants. Overall, the results of our study indicate that the developmental stage of the host plant is an important factor of conditionality in the interaction between C. brasiliense and arboreal foraging ants.     

Evaluating neurophylogenetic patterns in the larval nervous systems of brachiopods and their evolutionary significance to other bilaterian phyla

Recent structural analyses of invertebrate nervous systems have supported hypotheses stating that specific developmental and cytological aspects of larval and adult brains are conserved among bilaterian animals. Opposing views argue that structural similarities in larval nervous systems may be the result of convergent evolution and that the developmental diversity of adult brains is more indicative of several independent origins. Here, I use various cytological probes, confocal microscopy, and reconstruction techniques to investigate the cellular diversity within the larval nervous systems of Glottidia pyramidata and Terebratalia transversa (Brachiopoda). Neuronal cell types are compared among the rhynchonelliform, linguliform, and craniiform brachiopods as well as the phoronids. Although the respective larval types of the previously mentioned systematic groups clearly diverge in the neuroarchitecture of their larval apical organs (and nervous systems in general), a ground plan is proposed based on shared, centrally‐located, peptidergic neuronal cell types that can be compared with similar cell types in other lophotrochozoan phyla (bryozoans and spiralians). Assessing hierarchal levels of homology within and among the nervous systems of morphologically disparate phyla is challenging in that many phyla share early developmental signals that induce the specification of the neural ectoderm, clouding our ability to discern divergent larval and juvenile brain structure. Solving these problems will require a combined effort involving both traditional and more recent cytological techniques with a diversity of molecular probes that will better map the neuronal complexity of diverse invertebrate nervous systems. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Volume and density of microglomeruli in the honey bee mushroom bodies do not predict performance on a foraging task

The mushroom bodies (MBs) are insect brain regions important for sensory integration, learning, and memory. In adult worker honey bees (Apis mellifera ), the volume of neuropil associated with the MBs is larger in experienced foragers compared with hive bees and less experienced foragers. In addition, the characteristic synaptic structures of the calycal neuropils, the microglomeruli, are larger but present at lower density in 35‐day‐old foragers relative to 1‐day‐old workers. Age‐ and experience‐based changes in plasticity of the MBs are assumed to support performance of challenging tasks, but the behavioral consequences of brain plasticity in insects are rarely examined. In this study, foragers were recruited from a field hive to a patch comprising two colors of otherwise identical artificial flowers. Flowers of one color contained a sucrose reward mimicking nectar; flowers of the second were empty. Task difficulty was adjusted by changing flower colors according to the principle of honey bee color vision space. Microglomerular volume and density in the lip (olfactory inputs) and collar (visual inputs) compartments of the MB calyces were analyzed using anti‐synapsin I immunolabeling and laser scanning confocal microscopy. Foragers displayed significant variation in microglomerular volume and density, but no correlation was found between these synaptic attributes and foraging performance. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1057–1071, 2017     

How do groups of red imported fire ants (Hymenoptera: Formicidae) feed on a droplet of sugar water?

Many previous studies have focused on the foraging behaviors and strategies of the red imported fire ants, Solenopsis invicta Buren on solid food or granular bait; little attention has been paid to how liquid sugar is fed upon. In the present study, behavioral responses of S. invicta to 25% sucrose water droplets were observed. Five foraging patterns were identified in S. invicta colonies under laboratory conditions: (i) no feeding, no sucrose water feeding was observed; (ii) surround feeding, ants surrounded and fed along the edge of the sucrose droplet; (iii) stacked feeding, ants stacked and fed along the edge of the sucrose droplet; (iv) droplet‐break feeding, ants broke the liquid droplet and sucked sucrose water that spread on surface of the substance or soil particles previously transported by ants; and (v) cover feeding, whole surface of the sucrose droplet was covered by layers of feeding ants. This is the first time cover feeding in S. invicta has been reported, which obviously requires more ants compared to the other patterns. In addition, individual ants were tracked in videos under laboratory conditions, and behavioral repertoires that led to stacking, covering and droplet‐breaking were identified and described. The field investigation showed that surround feeding was most frequently performed by S. invicta foragers; however, cover feeding was not observed under field conditions during this study. Both laboratory and field studies showed colony‐level variations in sugar‐water feeding.     

Biogenic amines in the ponerine ant Harpegnathos saltator: serotonin and dopamine immunoreactivity in the brain

Many studies suggest a role for biogenic amines in a variety of insect behaviors including intraspecific aggression. In ants, despite a rich behavioral repertoire and prominent aggressive interactions, little is known about the potential impact of biogenic amines. This may partly be due to the general lack of information about aminergic systems in the ant brain. The present study investigates serotonergic and dopaminergic neuronal systems in the brain of the ponerine ant Harpegnathos saltator. In H. saltator, intraspecific aggression is important for the regulation of reproduction. This species, therefore, is amenable to comparative studies of aminergic neuronal effects on long-term changes in aggression. Using immunocytochemistry and confocal microscopy, we found that in the brains of sterile workers, the distributions of serotonergic and dopaminergic neuronal processes differed substantially. In addition, branching patterns of serotonergic neurons showed marked differences between males and females. Brains of workers after 3 days and 3 weeks of aggressive interactions revealed no marked differences in serotonergic and dopaminergic neurons compared to those of reproductive and non-aggressive individuals. We conclude that different levels of intraspecific aggression do not involve profound anatomical changes in serotonergic and dopaminergic neurons. Subtle changes may be masked by inter-individual variances.     

Drosophila growth cones: A genetically tractable platform for the analysis of axonal growth dynamics

The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F‐actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross‐talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F‐actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin‐MT linking factor Short stop, thus identifying an essential molecular player in this context. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2010