Roger C. Hardie

Significance: To move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes’ optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes’ whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies’ depth-perception dynamics, limits, and visual behaviors.

Neural mechanisms behind stereopsis, which requires simultaneous disparity inputs from two eyes, have remained mysterious. Here we show how ultrafast mirror-symmetric photomechanical contractions in the frontal forward-facing left and right eye photoreceptors give Drosophila super-resolution 3D-vision. By interlinking multiscale in vivo assays with multiscale simulations, we reveal how these photoreceptor microsaccades - by verging, diverging and narrowing the eyes’ overlapping receptive fields - channel depth information, as phasic binocular image motion disparity signals in time. We further show how peripherally, outside stereopsis, microsaccadic sampling tracks a flying fly’s optic flow field to better resolve the world in motion. These results change our understanding of how insect compound eyes work and suggest a general dynamic stereo-information sampling strategy for animals, robots and sensors. Significance statement: To move efficiently, animals must continuously work out their x,y,z-positions in respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes’ optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes’ whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a new morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila super-resolution 3D-vision and proposes neural computations for accurately predicting these flies’ depth-perception dynamics, limits, and visual behaviors.

Phototransduction inDrosophilais mediated by phospholipase C (PLC) and Ca²⁺-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca²⁺stores in this important model for Ca²⁺signaling remains obscure. We therefore expressed a low affinity Ca²⁺indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia andin vivofrom intact flies of both sexes. Blue excitation light induced a rapid (tau ∼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca²⁺stores, followed by a slower decay, typically reaching ∼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100–200 s. Both rapid and slow store depletion were largely unaffected in InsP₃receptor mutants, but were much reduced intrpmutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na⁺and in mutants of the Na⁺/Ca²⁺exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression ofcalxgreatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na⁺/Ca²⁺exchange across the ER membrane induced by Na⁺influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na⁺/Ca²⁺exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca²⁺in photoreceptors exposed to Ca²⁺-free solutions.SIGNIFICANCE STATEMENTPhototransduction inDrosophilais mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca²⁺stores play any role. We therefore engineered flies expressing a genetically encoded Ca²⁺indicator in the photoreceptor ER. Although NCX Na⁺/Ca²⁺exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca²⁺stores mediated by Na⁺/Ca²⁺exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca²⁺in photoreceptors bathed in Ca²⁺-free solutions.

Ca²⁺-activated K⁺ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modeling, we discover how visual information transfer in mutants lacking the BK channel (dSlo⁻), SK channel (dSK⁻), or both (dSK⁻;; dSlo⁻) is shaped in the female fruit fly (Drosophila melanogaster) R1–R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1–R6 Shaker and Shab K⁺ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants9 distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca²⁺-activated K⁺ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information. SIGNIFICANCE STATEMENT In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.

Ca ²⁺ -activated K ⁺ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modelling, we discover how visual information transfer in mutants lacking the BK channel ( dSlo ⁻ ), SK channel ( dSK ⁻ ) or both ( dSK ⁻ ;; dSlo ⁻ ) is shaped in the female fruit fly ( Drosophila melanogaster ) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K ⁺ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants’ distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca ²⁺ -activated K ⁺ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information. Significance statement: In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.

Phototransduction in Drosophila is mediated by phospholipase C-dependent hydrolysis of PIP2­, and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in Drosophila photoreceptors. Here, we used transgenic flies expressing fluorescently tagged probes (P4M and TbR332H), which allow in vivo quantitative measurements of PI4P and PIP2 dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, we identified Drosophila PI4KIIIα (CG10260) as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. Our results also indicate that PI4KIIIα activity requires rbo (the Drosophila orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIα activity in yeast and mammals. However, our evidence indicates that the recently reported central role of dPIP5K59B (CG3682) in PIP2 synthesis in the rhabdomeres should be re-evaluated; although PIP2 resynthesis was suppressed by RNAi directed against dPIP5K59B, little or no defect was detected in a reportedly null mutant (dPIP5K18).

Small fly eyes should not see fine image details. Because flies exhibit saccadic visual behaviors and their compound eyes have relatively few ommatidia (sampling points), their photoreceptors would be expected to generate blurry and coarse retinal images of the world. Here we demonstrate that Drosophila see the world far better than predicted from the classic theories. By using electrophysiological, optical and behavioral assays, we found that R1-R6 photoreceptors’ encoding capacity in time is maximized to fast high-contrast bursts, which resemble their light input during saccadic behaviors. Whilst over space, R1-R6s resolve moving objects at saccadic speeds beyond the predicted motion-blur-limit. Our results show how refractory phototransduction and rapid photomechanical photoreceptor contractions jointly sharpen retinal images of moving objects in space-time, enabling hyperacute vision, and explain how such microsaccadic information sampling exceeds the compound eyes’ optical limits. These discoveries elucidate how acuity depends upon photoreceptor function and eye movements.

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e. spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.