Biology subjects

Ispizua, J. I.

Publications and source records attributed to Ispizua, J. I..

7 recordsLinked to original sources

bioRxiv2026

Neural control of respiration in Drosophila

Adaptive control of breathing is essential for life, yet the neural circuits that couple respiration to locomotion and internal physiological state remain poorly understood. Insects regulate gas exchange through spiracles: valve-like openings in the cuticle that allow oxygen uptake while minimizing water loss. We find that spiracle opening in Drosophila is dynamically matched to flight power and reduced by dehydration. We identify the motor neurons that innervate the spiracle muscles and show that their optogenetic activation closes all eighteen spiracles and rapidly limits flight power. Inhibitory interneurons transmit descending flight commands to the spiracle motor neurons, opening spiracles in proportion to metabolic demand. A parallel interoceptive pathway converges on the same interneurons to close the spiracles and suppress flight. Feedforward descending commands thus couple flight power to spiracle opening, while interoceptive feedback pathways close spiracles to balance oxygen supply against water loss. This compact circuit architecture may reflect a general solution for matching respiration to the competing demands of locomotion and water conservation.

neuroscience↗

bioRxiv2026

Whole-body 3D kinematics of freely behaving Drosophila

Understanding how nervous systems generate coordinated movement requires precise measurement of body kinematics during natural behavior. The fruit fly, Drosophila, is a model organism with sophisticated behavior and well-studied neural circuits, but tracking fly movements in 3D remains challenging because of their teeny bodies, rapid movements, and frequent self-occlusions. Here we present a pipeline for markerless, full-body 3D pose estimation of fly terrestrial behavior, combining seven synchronized high-speed cameras to capture whole-body kinematics at 800 frames per second. We trained a hybrid 2D/3D deep learning model to track 50 keypoints, then refined them to produce anatomically feasible kinematic trajectories through a retargeting process that solved an inverse kinematics problem constrained by a biomechanical body model. Analysis of 3D kinematics revealed that flies perform grounded running across their full speed range, without transitioning between discrete gaits. Using multi-animal tracking, we found that courting males coordinate both wings during song and modulate body pitch to track the female's vertical position. Our open-source pipeline and large 3D kinematic dataset of fly behavior provide a foundation for neuromechanical modeling and mechanistic studies of motor control in a genetically tractable model organism.

neuroscience↗

bioRxiv2026

Adapting Upright Light Sheet Fluorescence Microscopy for Imaging at Air-Liquid Interface

Light sheet fluorescence microscopy (LSFM) is increasingly appreciated as the gold standard for gentle, volumetric imaging with fast acquisition speeds and/or long imaging durations. However, the often-constrained sample space of these microscopes has precluded a specific class of biological specimens from being studied with these tools: those requiring an air-liquid interface (ALI). Here, we present a device for robust imaging at ALI on an upright light sheet microscope with dipping objectives. We demonstrate the system using three relevant use-cases: ex vivo embryonic mouse salivary glands, human epidermal equivalent cultures, and in vivo adult Drosophila melanogaster brains. While the device presented is engineered for one specific light sheet microscope design, it provides a blueprint for easy adaptation to other systems. In doing so, it can potentially spur the use of LSFM for model systems that have so far been unable to take advantage of this powerful technology.

bioengineering↗

bioRxiv2026

Assessment of adult structural plasticity in Drosophila neurons

Unraveling how adult neurons reshape their architecture is key to understanding post-developmental plasticity. Drosophila clock neurons, which remodel their terminals on a daily basis, offer a unique model to examine the mechanisms underlying structural plasticity. In this study, we examine the impact of the experimental design on the remodeling process. We established a simple fixation protocol that preserves tissue integrity and prevents its deformation while enabling the fixation of a larger number of individuals within the appropriate time window. We show that intrinsic (i.e., targeting fluorescent reporters to the membrane) or extrinsic (i.e., temperature) variables may influence this dynamic process. Examining ex vivo preparations, we found that the s-LNv terminals display numerous thin filopodia extending from their synaptic boutons. However, these fine membrane protrusions are lost upon fixation, as they could only be accurately visualized ex vivo. Finally, we present MorphoScope, a Python-based interface that eliminates observer bias in complexity measurements. Altogether, we present a powerful and robust model to investigate the principles of adult neuronal plasticity, with implications extending beyond circadian biology.

neuroscience↗

bioRxiv2024

Single Objective Light Sheet Microscopy allows high-resolution in vivo brain imaging of Drosophila.

In vivo imaging of dynamic sub-cellular brain structures in Drosophila melanogaster is key to understanding several phenomena in neuroscience. However, its implementation has been hindered by a trade-off between spatial resolution, speed, photobleaching, phototoxicity, and setup complexity required to access the specific target regions of the small brain of Drosophila. Here, we present a single objective light-sheet microscope, customized for in vivo imaging of adult flies and optimized for maximum resolution. With it, we imaged the axonal projections of small lateral ventral neurons (known as s-LNvs) in intact adult flies. We imaged the plasma membrane, mitochondria, and dense-core vesicles with high spatial resolution up to 370 nm, ten times lower photobleaching than confocal microscopy, lower invasiveness and complexity in sample mounting than alternative light-sheet technologies, and without relying on phototoxic pulsed infrared lasers. This unique set of features paves the way for new long-term, dynamic studies in the brains of living flies.

neuroscience↗

bioRxiv2024

Ultrastructural correlates of circadian structural plasticity

In Drosophila, about 250 clock neurons in the brain form a network that orchestrates circadian rhythmicity. Among them, eight small Lateral ventral Neurons (s-LNvs) play a critical role, synchronizing the circadian ensemble via the neuropeptide Pigment-Dispersing Factor (PDF). Moreover, their neurites show daily variations in morphology, PDF levels, synaptic markers and connectivity. This process, called circadian structural plasticity, is ill-defined at the subcellular level. Here, we present 3D volumes of the s-LNv terminals generated by Serial Block-face Scanning Electron Microscopy (SBEM) at three key time points, two hours before lights-ON, two hours after lights-ON, and two hours after lights-OFF. We report a reduction in the number of neuronal varicosities at night, which reflects (and probably regulates) the cycling of the components we found therein. Indeed, in the morning we observed more presynaptic sites and increased accumulation and release of dense core vesicles. These rhythms were paralleled by periodic changes in mitochondrial structure that suggest daily modulation of their activity. We propose that circadian plasticity of the functionally relevant structures within presynaptic varicosities cyclically modulates the influence of the s-LNvs on the clock network.

neuroscience↗

bioRxiv2022

Mating disrupts morning anticipation in Drosophila melanogaster females 2

After mating, the physiology of Drosophila females undergoes several important changes, some of which are reflected in their rest-activity cycles. To explore the hypothesis that mating modifies the temporal organization of locomotor activity patterns, we recorded the fly activity by a video tracking method. Monitoring rest-activity patterns under light/dark (LD) cycles indicated that mated females lose their ability to anticipate the night-day transition, in stark contrast to males and virgins; this postmating response is mediated by the sex peptide (SP) acting mainly on pickpocket (ppk) expressing neurons, since reducing expression of the SP receptor (SPR) in these neurons restores the ability to anticipate the LD transition in mated females. We further provide evidence of connectivity between PPK+ neurons and the pigment-dispersing factor (PDF)-positive ventral lateral neurons (sLNv), which play a central role in the temporal organization of daily activity. Since PDF has been associated to the generation of the morning activity peak, we hypothesized that the mating signal could modulate PDF levels. Indeed, mated females have reduced PDF levels at the dorsal protocerebrum; moreover, SPR downregulation in PPK+ neurons mimics PDF levels observed in males. In sum, our results are consistent with a model whereby mating-triggered signals reaches clock neurons in the fly central nervous system to modulate the temporal organization of circadian behavior according to the needs of the new status. Author SummaryAfter mating, Drosophila females undergoes striking behavioral changes, specially in their activity patterns. Despite some of the circuits that deliver mating signals to the female brain are known the connection with the circadian network has not been explored in detail. Here, we show that mating changes the onset of daily activity, masking a central function of the clock. This modulation is mediated by the sex peptide (transferred during courtship) acting on PPK+ neurons, which, in turn, directly contact PDF+ neurons, responsible for the increase of the activity that precedes dawn. Thus, our work identifies a postmating response directly related to the circadian clock, and begins to unravel the underlying neuronal circuit.

neuroscience↗