Binocular mirror-symmetric microsaccadic sampling enables Drosophila hyperacute 3D vision.

SignificanceTo 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.

Wiring dendrites in layers and columns.

The most striking structure in the nervous system is the complex yet stereotyped morphology of the neuronal dendritic tree. Dendritic morphologies and the connections they make govern information flow and integration in the brain. The fundamental mechanisms that regulate dendritic outgrowth and branching are subjects of extensive study. In this review, we summarize recent advances in the molecular and cellular mechanisms for routing dendrites in layers and columns, prevalent organizational structures in the brain. We highlight how dendritic patterning influences the formation of synaptic circuits.

A gustatory second-order neuron that connects sucrose-sensitive primary neurons and a distinct region of the gnathal ganglion in the Drosophila brain.

Although the gustatory system provides animals with sensory cues important for food choice and other critical behaviors, little is known about neural circuitry immediately following gustatory sensory neurons (GSNs). Here, we identify and characterize a bilateral pair of gustatory second-order neurons (G2Ns) in Drosophila. Previous studies identified GSNs that relay taste information to distinct subregions of the primary gustatory center (PGC) in the gnathal ganglia (GNG). To identify candidate G2Ns, we screened ∼5,000 GAL4 driver strains for lines that label neural fibers innervating the PGC. We then combined GRASP (GFP reconstitution across synaptic partners) with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate G2Ns and the axonal terminals of Gr5a-expressing GSNs, which are known to respond to sucrose. Results of the GRASP analysis, followed by a single-cell analysis by FLP-out recombination, revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, we expressed fluorescence-tagged acetylcholine receptor subunit (Dα7) and active-zone marker (Brp) in the G2Ns. We found that G2N input sites overlaid GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons' arborizations. GRASP analysis and further tests with the Syb-GRASP method suggested that the identified G2Ns receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the GNG, and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). Our findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.

Multiple redundant medulla projection neurons mediate color vision in Drosophila.

The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies' optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.

Use of a Drosophila genome-wide conserved sequence database to identify functionally related cis-regulatory enhancers.

BACKGROUND: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. RESULTS: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. CONCLUSIONS: cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process.

Conserved alternative splicing and expression patterns of arthropod N-cadherin.

Metazoan development requires complex mechanisms to generate cells with diverse function. Alternative splicing of pre-mRNA not only expands proteomic diversity but also provides a means to regulate tissue-specific molecular expression. The N-Cadherin gene in Drosophila contains three pairs of mutually-exclusive alternatively-spliced exons (MEs). However, no significant differences among the resulting protein isoforms have been successfully demonstrated in vivo. Furthermore, while the N-Cadherin gene products exhibit a complex spatiotemporal expression pattern within embryos, its underlying mechanisms and significance remain unknown. Here, we present results that suggest a critical role for alternative splicing in producing a crucial and reproducible complexity in the expression pattern of arthropod N-Cadherin. We demonstrate that the arthropod N-Cadherin gene has maintained the three sets of MEs for over 400 million years using in silico and in vivo approaches. Expression of isoforms derived from these MEs receives precise spatiotemporal control critical during development. Both Drosophila and Tribolium use ME-13a and ME-13b in "neural" and "mesodermal" splice variants, respectively. As proteins, either ME-13a- or ME-13b-containing isoform can cell-autonomously rescue the embryonic lethality caused by genetic loss of N-Cadherin. Ectopic muscle expression of either isoform beyond the time it normally ceases leads to paralysis and lethality. Together, our results offer an example of well-conserved alternative splicing increasing cellular diversity in metazoans.

Functional Imaging from Fly Photoreceptors.

In this protocol, we describe the procedures for visualizing light-evoked calcium changes in fly visual neurons using two-photon microscopy. Before starting the imaging, the visual stimulation system should be set up properly. To facilitate later data analysis, we recommend synchronizing (or time-stamping) imaging and visual stimuli during experiments. Depending on the scientific question and experimental design, the visual stimuli can be modified. Here we provide an example protocol for measuring the intensity-response function in fly ultraviolet (UV)-sensing photoreceptors using UV illumination. For this purpose, precise time-stamping or synchronization is not required.

Dissection of the Head of a Live Fly for Functional Imaging.

In this protocol, we outline procedures to mount the fly and to open up the head cuticle to expose the optic lobes for in vivo imaging. The fly is first inserted into a custom-made fly chamber in which the fly's head is stabilized on a piece of aluminum foil. Once the fly is mounted in the chamber, its head cuticle is removed, exposing the optic lobe for recording. The brain tissues (above the foil), including the optic lobes, should be bathed in fly saline. Meanwhile, the eyes (below the foil) are kept dry to receive light stimuli during the recording. A considerable level of expertise and hand dexterity is required to handle a small animal such as a fly, especially when opening its head capsule without damaging the brain tissue. This expertise should be gained through mindful repetition of the protocol. With appropriate preparation and skills, the success rate for this procedure can be >95%. Using this protocol, it is possible to record ultraviolet (UV)-sensing photoreceptors, which have long visual fibers that terminate at the medulla (the second optic neuropil). Depending on the visual neurons of interest, some modifications to fly mounting might be needed.

Calcium Imaging of Neural Activity in Fly Photoreceptors.

Functional imaging methodologies allow researchers to simultaneously monitor the neural activities of all single neurons in a population, and this ability has led to great advances in neuroscience research. Taking advantage of a genetically tractable model organism, functional imaging in Drosophila provides opportunities to probe scientific questions that were previously unanswerable by electrophysiological recordings. Here, we introduce comprehensive protocols for two-photon calcium imaging in fly visual neurons. We also discuss some challenges in applying optical imaging techniques to study visual systems and consider the best practices for making comparisons between different neuron groups.

Quantitative Analysis of Photoreceptor Intensity-Response Function in Fly Visual Neurons.

In this protocol, we illustrate how to process images acquired during functional imaging of fly visual neurons and how to analyze and quantify visually evoked activities. We use ImageJ/Fiji for the initial imaging processing. All images acquired previously should be registered to compensate for tissue movement. Next, we extract fluorescence signals specifically from neurons that respond to the light by marking the regions of interest (ROIs). The data are further analyzed in a data-analysis program, such as MATLAB, to plot response traces against time. Finally, we obtain different parameters to reveal the neuron's physiological properties by fitting the data with a Naka-Rushton function.

Tiling of r7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion.

The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. We refer to the restriction of terminals to single columns as tiling. In a genetic screen based on an R7-dependent behavior, we identified the Activin receptor Baboon and the nuclear import adaptor Importin-alpha3 as being required to prevent R7 axon terminals from overlapping with the terminals of R7s in neighboring columns. This tiling function requires the Baboon ligand, dActivin, the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus. We propose that dActivin is an autocrine signal that restricts R7 growth cone motility, and we demonstrate that it acts in parallel with a paracrine signal that mediates repulsion between R7 terminals.

Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster.

In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. Here, we genetically dissect the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. We now show that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior.

Two-photon scanned light sheet fluorescence microscopy with axicon imaging for fast volumetric imaging.

SIGNIFICANCE: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition. AIM: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM. APPROACH: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM. RESULTS: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz. CONCLUSIONS: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.

Neural mechanism of spatio-chromatic opponency in the Drosophila amacrine neurons.

Visual animals detect spatial variations of light intensity and wavelength composition. Opponent coding is a common strategy for reducing information redundancy. Neurons equipped with both spatial and spectral opponency have been identified in vertebrates but not yet in insects. The Drosophila amacrine neuron Dm8 was recently reported to show color opponency. Here, we demonstrate Dm8 exhibits spatio-chromatic opponency. Antagonistic convergence of the direct input from the UV-sensing R7s and indirect input from the broadband receptors R1-R6 through Tm3 and Mi1 is sufficient to confer Dm8's UV/Vis (ultraviolet/visible light) opponency. Using high resolution monochromatic stimuli, we show the pale and yellow subtypes of Dm8s, inheriting retinal mosaic characteristics, have distinct spectral tuning properties. Using 2D white-noise stimulus and reverse correlation analysis, we found that the UV receptive field (RF) of Dm8 has a center-inhibition/surround-excitation structure. In the absence of UV-sensing R7 inputs, the polarity of the RF is inverted owing to the excitatory input from the broadband photoreceptors R1-R6. Using a new synGRASP method based on endogenous neurotransmitter receptors, we show that neighboring Dm8s form mutual inhibitory connections mediated by the glutamate-gated chloride channel GluClα, which is essential for both Dm8's spatial opponency and animals' phototactic behavior. Our study shows spatio-chromatic opponency could arise in the early visual stage, suggesting a common information processing strategy in both invertebrates and vertebrates.

Extrinsic Factors Regulating Dendritic Patterning.

Stereotypic dendrite arborizations are key morphological features of neuronal identity, as the size, shape and location of dendritic trees determine the synaptic input fields and how information is integrated within developed neural circuits. In this review, we focus on the actions of extrinsic intercellular communication factors and their effects on intrinsic developmental processes that lead to dendrite patterning. Surrounding neurons or supporting cells express adhesion receptors and secreted proteins that respectively, act via direct contact or over short distances to shape, size, and localize dendrites during specific developmental stages. The different ligand-receptor interactions and downstream signaling events appear to direct dendrite morphogenesis by converging on two categorical mechanisms: local cytoskeletal and adhesion modulation and global transcriptional regulation of key dendritic growth components, such as lipid synthesis enzymes. Recent work has begun to uncover how the coordinated signaling of multiple extrinsic factors promotes complexity in dendritic trees and ensures robust dendritic patterning.

Antagonistic regulation by insulin-like peptide and activin ensures the elaboration of appropriate dendritic field sizes of amacrine neurons.

Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. Here we report that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. We found DILP2 was expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. We suggest that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields.

Neto-α Controls Synapse Organization and Homeostasis at the Drosophila Neuromuscular Junction.

Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

Visual circuit development in Drosophila.

Fly visual circuits are organized into lattice-like arrays and layers. Recent genetic studies have provided insights into how these reiterated structures are assembled through stepwise processes and how precise connections are established during development. Afferent-derived morphogens, such as Hedgehog, play a key role in organizing the overall structure by inducing and recruiting target neurons and glia. In turn, the target-derived ligand DWnt4 guides Frizzled2-expressing photoreceptor afferents to their proper destination. Photoreceptor afferents select specific synaptic targets by forming adhesive interactions and regulating actin cytoskeleton in growth cones. Target specificity is probably achieved by restricting the expression of adhesive molecules, such as Capricious, to appropriate presynaptic and postsynaptic partners, and by differentially regulating the function of broadly expressed adhesive molecules such as N-cadherin.