Ezequiel Axel Gorostiza

Summary: Walking animals must continuously reorient to navigate their environment, yet how leg kinematics are reconfigured to produce directional changes of varying magnitude remains poorly understood. Here, we used high-resolution leg tracking to characterize steering behavior in freely walking Drosophila . We focus our analysis on two major strategies, arcs and turns. Although arc and turn trajectories are statistically separable, several parameters suggest an underlying spatial continuum spanning both behavioral categories, involving a change in the steering mechanism rather than a discontinuity in the kinematic space. Arcs, representing shallow curves, and were generated primarily through: (1) graded shifts in the posterior extreme position (PEPs) of the front and inside middle legs, and bilateral asymmetries in stance amplitude, lengthening on the outside (2) and shortening on the inside (3) while step timing and ipsilateral coordination remained at straight-walking baseline. Turns, in contrast, were associated with higher steering demands and relied on: (4) progressive displacement of the anterior extreme positions (AEPs) in front and middle legs, (5) extreme reduction of inside middle and hind leg amplitudes to anchor the turning pivot, (6) skipped steps of inside middle and hind legs, (7) lateral steps of inside middle legs, and (8) spontaneous appearance of synchronized backward-stepping in particular legs beyond a critical turning threshold. Finally, a common kinematic configuration emerges at maximal turning demand. These results suggest a hierarchical, bipartite organization of steering: PEP- and AEP-driven mechanisms operating across moderate and severe trajectory curvatures, and a shared saturated motor state at the behavioral extreme that links descending steering commands to segment-specific leg kinematics in a freely walking insect Graphical Highlights: Drosophila flies use several strategies to change direction, including arcs and turns Arcs have constant curvature, speed and constant angular velocity, while turns require a decrease in speed before changing direction and their angular velocity tends to be higher. We identified eight leg kinematic mechanisms that contribute to these strategies. Some kinematic parameters vary smoothly and in a continuous fashion between mechanisms.

Summary: With the newly uncovered access to a genetic line labeling all campaniform sensilla in Drosophila melanogaster and the many insights gathered from larger insects, we analyzed the distribution and patterning of this class of proprioceptors throughout the fly nervous system and studied CS function in motor control. We demonstrate via two-photon calcium imaging microscopy that campaniform sensilla activation induces activity in many leg muscles, showing that these proprioceptive stimuli can influence motor neuron activity. We then dissected how the lack of these proprioceptive stimuli influence walking behavior, leg kinematics, and interleg coordination in freely-walking flies using transient optogenetic inhibition and video tracking with high spatiotemporal resolution. We show that campaniform sensilla inhibition robustly affects Drosophila melanogaster ’s ability to reach their typical walking speeds. Detailed analysis of leg kinematic suggests that shorter stance amplitudes characterized by their long-lasting duration were behind the speed deficits. Dissecting this phenotype also revealed the inability to maintain coordinated interleg stepping patterns as well as postural control in absence of proprioceptive force and load feedback.

The actin/spectrin membrane-associated periodic skeleton (MPS) is a ubiquitous cytoskeletal structure essential for axonal integrity and function. Primarily studied in cultured neurons, the MPS has been extensively modeled as actin rings spaced by spectrin tetramers, the latter assumed to be regularly and densely distributed across the axonal perimeter. However, its nanoscale organization within native tissue environments remains poorly understood. In this study, we investigated the three-dimensional organization of βII-spectrin in the mouse sciatic nerve using 3D-dSTORM and STED super-resolution microscopy on thin transversal cryosections. By implementing a custom quantitative analysis pipeline, we resolved the sub-diffraction architecture of the MPS across myelinated axons of diverse diameters. We confirm that βII-spectrin is localized to the inner face of the axonal plasma membrane and maintains a longitudinal periodicity of approximately 170 nm, consistent with previous observations. Crucially, 3D-dSTORM revealed that βII-spectrin along the axonal perimeter is organized in discrete nanoscale clusters with a median effective radius of 25 nm, compatible with the size of an individual spectrin tetramer visualized by indirect immunolabeling. The number of these clusters scales linearly with the axonal perimeter, maintaining a constant membrane occupancy of ∼20% across varying axon diameters. Moreover, these clusters exhibit a non-random spatial distribution with a characteristic center-to-center nearest-neighbor distance of ∼200 nm. These findings challenge simplified models of the MPS based on cultured systems and demonstrate that the MPS in peripheral nerves is composed of discrete structural units. This modular, dispersed organization may provide the structural flexibility required to withstand the mechanical demands of the peripheral nervous system while maintaining a stable periodic scaffold.

Summary: Animals need to fine-control the speed and direction of locomotion to navigate complex and dynamic environments. To achieve this, they integrate multimodal sensory inputs with their internal drive to constantly adjust their motor output. This integration involves the interplay of neuronal populations across different hierarchical levels along the sensorimotor axis – from sensory, central, and modulatory neurons in the brain to descending neurons and motor networks in the nerve cord. Here, we characterize two populations of neurons that control distinct aspects of walking on different hierarchical levels in Drosophila . First, we use in-vivo electrophysiological recordings to demonstrate that moonwalker descending neurons (MDN) integrate antennal touch to drive changes in walking direction from forward to backward. Second, we establish DopaMeander as an important component in the control of forward walking through a combination of optogenetic activation, silencing, connectomics, and in-vivo recordings. These dopaminergic modulatory neurons drive forward walking with increased turning, and the activity of individual neurons is correlated with ipsiversive turning. Hence, MDN and DopaMeander control opposite regimes of walking on different hierarchical levels. Computational models reveal that their activity predicts key parameters of spontaneous walking. Moreover, we find that both MDN and DopaMeander are gated out during flight. This suggests that neuronal populations across levels of control are modulated by the behavioral state to minimize cross-talk between motor programs.

The actin/spectrin membrane-associated periodic skeleton (MPS) is a cytoskeletal structure that supports axonal integrity and function. Lower spinal motor neurons (MNs) are characterized by exceptionally long axons and are particularly susceptible to degeneration in a wide range of hereditary neuromuscular disorders, including amyotrophic lateral sclerosis. Using confocal and super-resolution imaging, we characterized the spatial distribution βII-spectrin and the assembly pattern of the MPS in human MN axons derived from induced pluripotent stem cells (iPSCs). We discovered a striking gap-and-patch pattern in the medial axon, where sharply demarcated βII-spectrin gaps alternate with patches containing a well-organized MPS. The pattern is acutely induced by the kinase inhibitor staurosporine and pharmacological inhibition of actin polymerization prevents patch formation, indicating a requirement for actin nucleation in MPS assembly. Our data supports a model in which spectrin incorporation into nascent MPS patches depletes neighboring regions, producing long-range gaps-and-patches patterns.

Our own unique character traits make our behavior consistent and define our individuality. Yet, this consistency does not entail that we behave repetitively like machines. Like humans, animals also combine personality traits with spontaneity to produce adaptive behavior: consistent, but not fully predictable. Here, we study an iconically rigid behavioral trait, insect phototaxis, that nevertheless also contains both components of individuality and spontaneity. In a light/dark T-maze, approximately 70% of a group of Drosophila fruit flies choose the bright arm of the T-Maze, while the remaining 30% walk into the dark. Taking the photopositive and the photonegative subgroups and re-testing them reveals the spontaneous component: a similar 70–30 distribution emerges in each of the two subgroups. Increasing the number of choices to ten choices, reveals the individuality component: flies with an extremely negative series of first choices were more likely to show photonegative behavior in subsequent choices and vice versa. General behavioral traits, independent of light/dark preference, contributed to the development of this individuality. The interaction of individuality and spontaneity together explains why group averages, even for such seemingly stereotypical behaviors, are poor predictors of individual choices.

Our own unique character traits make our behavior consistent and define our individuality. Yet, this consistency does not entail that we behave repetitively like machines. Like humans, animals also combine personality traits with spontaneity to produce adaptive behavior: consistent, but not fully predictable. Here, we study an iconically rigid behavioral trait – insect phototaxis – that that nevertheless also contains both components of individuality and spontaneity. In a light/dark T-maze, approximately 70% of a group of Drosophila fruit flies choose the bright arm of the T-Maze, while the remaining 30% walk into the dark. Taking the photopositive and the photonegative subgroups and re-testing them reveals the spontaneous component: a similar 70-30 distribution emerges in each of the two subgroups. Increasing the number of choices to ten choices, reveals the individuality component: flies with extremely negative first choices were more likely to show photonegative behavior in subsequent choices and vice versa . General behavioral traits, independent of light/dark preference, contributed to the development of this individuality. The interaction of individuality and spontaneity together explains why group averages, even for such seemingly stereotypical behaviors, are poor predictors of individual choices.

Does Cognition Have a Role in Plasticity of “Innate Behavior”? A Perspective From Drosophila

Science and education are key factors in the successful development of countries, and those that actively support both have better chances to improve their general conditions. However, the budgets destined to these areas in developed and developing countries cannot be matched, even with the best intentions of a government, and the gap between them has uncountable consequences. As an example, a lower budget hinders the ability to acquire state-of-the-art equipment, which affects the possibilities to catch up with the latest questions in research, and decrease the competitivity of developing countries for international grants, further increasing the gap. In this article, the authors provide instructions to build a cheap but highly versatile open-source platform for basic fluorescence microscopy, optogenetics, and temperature control behavioural experiments. The platform is designed to work with zebrafish, Drosophila, and Caenorhabditis elegans. The low cost of the parts (200 euros in total, without counting the 3D printer required) and the design make this an affordable and excellent tool, not only for teaching at universities and schools in the developing world but also for research and diagnosis. It is always important to recognize the effort made by the people who try to increase science literacy around the world, and decrease the differences between countries. Moreover, these works need as much publicity as possible to reach their goal. That is why we recommend this article.