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Color vision is an important sensory capability that enhances the detection of contrast in retinal images. Monochromatic animals exclusively detect temporal and spatial changes in luminance, whereas two or more types of photoreceptors and neuronal circuitries for the comparison of their responses enable animals to differentiate spectral information independent of intensity. Much of what we know about the cellular and physiological mechanisms underlying color vision comes from research on vertebrates including primates. In insects, many important discoveries have been made, but direct insights into the physiology and circuit implementation of color vision are still limited. Recent advances inDrosophilasystems neuroscience suggest that a complete insect color vision circuitry, from photoreceptors to behavior, including all elements and computations, can be revealed in future. Here, we review fundamental concepts in color vision alongside our current understanding of the neuronal basis of color vision inDrosophila,including side views to selected other insects.

Color vision is an important sensory capability of humans and many animals. It relies on color opponent processing in visual circuits that gradually compare the signals of photoreceptors with different spectral sensitivities. In Drosophila, this comparison begins already in the presynaptic terminals of UV-sensitive R7 and longer wavelength-sensitive R8 inner photoreceptors that inhibit each other in the medulla. How downstream neurons process their signals is unknown. Here, we report that the second order medulla interneuron Dm8 is inhibited when flies are stimulated with UV light and strongly excited in response to a broad range of longer wavelength (VIS) stimuli. Inhibition to UV light is mediated by histaminergic input from R7 and expression of the histamine receptor ort in Dm8, as previously suggested. However, two additional excitatory inputs antagonize the R7 input. First, activation of R8 leads to excitation of Dm8 by non-canonical photoreceptor signaling and cholinergic neurotransmission in the visual circuitry. Second, activation of outer photoreceptors R1-R6 with broad spectral sensitivity causes excitation in Dm8 through the cholinergic medulla interneuron Mi1, which is known for its major contribution to the detection of spatial luminance contrast and visual motion. In summary, Dm8 mediates a second step in UV/VIS color opponent processing in Drosophila by integrating input from all types of photoreceptors. Our results demonstrate novel insights into the circuit integration of R1-R6 into color opponent processing and reveal that chromatic and achromatic circuitries of the fly visual system interact more extensively than previously thought.

Color vision extracts spectral information by comparing signals from photoreceptors with different visual pigments. Such comparisons are encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. Here, we examine the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. We report that coloropponent processing of UVshort/blue and UVlong/green is already implemented in R7/R8 inner photoreceptor terminals of "pale'' and "yellow'' ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently.

In the visual system of Drosophila, photoreceptors R1-R6 relay achromatic brightness information to five parallel pathways. Two of them, the lamina monopolar cells L1 and L2, represent the major input lines to the motion detection circuitry. We devised a new method for optical recording of visually evoked changes in intracellular Ca2+ in neurons using targeted expression of a genetically encoded Ca2+ indicator. Ca2+ in single terminals of L2 neurons in the medulla carried no information about the direction of motion. However, we found that brightness decrements (light-OFF) induced a strong increase in intracellular Ca2+ but brightness increments (light-ON) induced only small changes, suggesting that half-wave rectification of the input signal occurs. Thus, L2 predominantly transmits brightness decrements to downstream circuits that then compute the direction of image motion.

The nervous system of seeing animals derives information about optic flow in two subsequent steps. First, local motion vectors are calculated from moving retinal images, and second, the spatial distribution of these vectors is analyzed on the dendrites of large downstream neurons. In dipteran flies, this second step relies on a set of motion-sensitive lobula plate tangential cells (LPTCs), which have been studied in great detail in large fly species. Yet, studies on neurons that convey information to LPTCs and neuroanatomical investigations that enable a mechanistic understanding of the underlying dendritic computations in LPTCs are rare. We investigated the subcellular distribution of nicotinic acetylcholine receptors (nAChRs) on two sets of LPTCs: vertical system (VS) and horizontal system (HS) cells in Drosophila melanogaster. In this paper, we describe that both cell types express Dalpha7-type nAChR subunits specifically on higher order dendritic branches, similar to the expression of gamma aminobutyric acid (GABA) receptors. These findings support a model in which directional selectivity of LPTCs is achieved by the dendritic integration of excitatory, cholinergic, and inhibitory GABA-ergic input from local motion detectors with opposite preferred direction. Nonetheless, whole-cell recordings in mutant flies without Dalpha7 nAChRs revealed that direction selectivity of VS and HS cells is largely retained. In addition, mutant LPTCs were responsive to acetylcholine and remaining nAChR receptors were labeled by alpha-bungarotoxin. These results in LPTCs with genetically manipulated excitatory input synapses suggest a robust cellular implementation of dendritic processing that warrants direction selectivity. The underlying mechanism that ensures appropriate nAChR-mediated synaptic currents and the functional implications of separate sets or heteromultimeric nAChRs can now be addressed in this system.

At the Drosophila melanogaster larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30–100 boutons onto the muscle it innervates. How transmission strength is distributed among the boutons of the NMJ is unknown. To address this, we created synapcam, a version of the Ca2+ reporter Cameleon. Synapcam localizes to the postsynaptic terminal and selectively reports Ca2+ influx through glutamate receptors (GluRs) with single-impulse and single-bouton resolution. GluR-based Ca2+ signals were uniform within a given connection (that is, a given bouton/postsynaptic terminal pair) but differed considerably among connections of an NMJ. A steep gradient of transmission strength was observed along axonal branches, from weak proximal connections to strong distal ones. Presynaptic imaging showed a matching axonal gradient, with higher Ca2+ influx and exocytosis at distal boutons. The results suggest that transmission strength is mainly determined presynaptically at the level of individual boutons, possibly by one or more factors existing in a gradient. *Note: In the version of this article initially published online, the second author’s name was misspelled. The correct spelling should be Dierk F Reiff. The error has been corrected in the HTML version of the article. This correction has been appended to the PDF and print versions.

Genetically encoded fluorescent probes of neural activity represent new promising tools for systems neuroscience. Here, we present a comparativein vivoanalysis of 10 different genetically encoded calcium indicators, as well as the pH-sensitive synapto-pHluorin. We analyzed their fluorescence changes in presynaptic boutons of theDrosophilalarval neuromuscular junction. Robust neural activity did not result in any or noteworthy fluorescence changes when Flash-Pericam, Camgaroo-1, and Camgaroo-2 were expressed. However, calculated on the raw data, fractional fluorescence changes up to 18% were reported by synapto-pHluorin, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, GCaMP1.3, GCaMP1.6, and the troponin C-based calcium sensor TN-L15. The response characteristics of all of these indicators differed considerably from each other, with GCaMP1.6 reporting high rates of neural activity with the largest and fastest fluorescence changes. However, GCaMP1.6 suffered from photobleaching, whereas the fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible, with TN-L15 showing the fastest rise of the signals at lower activity rates. We show for GCaMP1.3 and YC3.3 that an expanded range of neural activity evoked fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR). The expression level of the indicator biased the signal kinetics and SNR, whereas the signal amplitude was independent. The presented data will be useful forin vivoexperiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of the optical signals.

In this study we established a transgenic Ca 2+ imaging technique in Drosophila that enabled us to target the Ca 2+ sensor protein yellow Cameleon-2 specifically to larval neurons. This noninvasive method allowed us to measure evoked Ca 2+ signals in presynaptic terminals of larval neuromuscular junctions (NMJs). We combined transgenic Ca 2+ imaging with electrophysiological recordings and morphological examinations of larval NMJs to analyze the mechanisms underlying persistently enhanced evoked vesicle release in two independent mutants. We show that persistent strengthening of junctional vesicle release relies on the recruitment of additional active zones, the spacing of which correlated with the evoked presynaptic Ca 2+ dynamics of individual presynaptic terminals. Knock-out mutants of the postsynaptic glutamate receptor (GluR) subunit DGluR-IIA, which showed a reduced quantal size, developed NMJs with a smaller number of presynaptic boutons but a strong compensatory increase in the density of active zones. This resulted in an increased evoked vesicle release on single action potentials and larger evoked Ca 2+ signals within individual boutons; however, the transmission of higher frequency stimuli was strongly depressed. A second mutant ( pabp P970 /+ ), which showed enhanced evoked vesicle release triggered by elevated subsynaptic protein synthesis, developed NMJs with an increased number of presynaptic boutons and active zones; however, the density of active zones was maintained at a value typical for wild-type animals. This resulted in wild-type evoked Ca 2+ signals but persistently strengthened junctional signal transmission. These data suggest that the consolidation of strengthened signal transmission relies not only on the recruitment of active zones but also on their equal distribution in newly grown boutons.

The developing neuromuscular junctions (NMJs) of Drosophila larvae can undergo long-term strengthening of signal transmission, a process that has been shown recently to involve local subsynaptic protein synthesis and that is associated with an elevated synaptic accumulation of the postsynaptic glutamate receptor subunit DGluR-IIA. To analyze the role of altered postsynaptic glutamate receptor expression during this form of genetically induced junctional plasticity, we manipulated the expression levels of two so far-described postsynaptic receptor subunit genes, dglur-IIA and dglur-IIB, in wild-type animals and plasticity mutants. Here we show that elevated synaptic expression of DGluR-IIA, which was achieved by direct transgenic overexpression, by genetically increased subsynaptic protein synthesis, or by a reduced dglur-IIB gene copy number, results in an increased recruitment of active zones, a corresponding enhancement in the strength of junctional signal transmission, and a correlated addition of boutons to the NMJ. Ultrastructural evidence demonstrates that active zones appear throughout NMJs at a typical density regardless of genotype, suggesting that the space requirements of active zones are responsible for the homogeneous synapse distribution and that this regulation results in the observed growth of additional boutons at strengthened NMJs. These phenotypes were suppressed by reduced or eliminated DGluR-IIA expression, which resulted from either a reduced dglur-IIA gene copy number or transgenic overexpression of DGluR-IIB. Our results demonstrate that persistent alterations of neuronal activity and subsynaptic translation result in an elevated synaptic accumulation of DGluR-IIA, which mediates the observed functional strengthening and morphological growth apparently through the recruitment of additional active zones.

The Journal of Neurogenetics presents this special issue to honor the lifelong contributions of Karl-Friedrich Fischbach on the occasion of his retirement from university duties in March 2014. Karl...

The optic lobe of Drosophila houses about 60,000 neurons that are organized in parallel, retinotopically arranged columns. Based on the Golgi-staining method, Fischbach and Dittrich ([1989] Cell Tissue Res 258:441-475) determined that each column contains about 90 identified cells. Each of these cells is supposed to release one or two different neurotransmitters. However, for most cells the released neurotransmitter is not known. Here we characterize the vast majority of the neurons in the Drosophila optic lobe that release acetylcholine (Ach), the major excitatory neurotransmitter of the insect central nervous system. We employed a promoter specific for cholinergic neurons and restricted its activity to single or a few cells using the MARCM technique. This approach allowed us to establish an anatomical map of neurons with a cholinergic phenotype based on their branching pattern. We identified 43 different types of neurons with a cholinergic phenotype. Thirty-one of them match previously described members of nine different subgroups: Transmedullary (Tm), Transmedullary Y (TmY), Medulla intrinsic (Mi, Mt, and Pm), Bushy T (T), Translobula Plate (Tlp), and Lobula intrinsic (Lcn and Lt) neurons (Fischbach and Dittrich [1989]). Intriguingly, 12 newly identified cell types suggest that previous Golgi studies were not saturating and that the actual number of different neurons per column is higher than previously thought. This study and similar ones on other neurotransmitter systems will contribute towards a columnar wiring diagram and foster the functional dissection of the visual circuitry in Drosophila.

Motion vision is essential for navigating through the environment. Due to its genetic amenability, the fruit fly Drosophila has been serving for a lengthy period as a model organism for studying optomotor behavior as elicited by large-field horizontal motion. However, the neurons underlying the control of this behavior have not been studied in Drosophila so far. Here we report the first whole cell recordings from three cells of the horizontal system (HSN, HSE, and HSS) in the lobula plate of Drosophila. All three HS cells are tuned to large-field horizontal motion in a direction-selective way; they become excited by front-to-back motion and inhibited by back-to-front motion in the ipsilateral field of view. The response properties of HS cells such as contrast and velocity dependence are in accordance with the correlation-type model of motion detection. Neurobiotin injection suggests extensive coupling among ipsilateral HS cells and additional coupling to tangential cells that have their dendrites in the contralateral hemisphere of the brain. This connectivity scheme accounts for the complex layout of their receptive fields and explains their sensitivity both to ipsilateral and to contralateral motion. Thus the main response properties of Drosophila HS cells are strikingly similar to the responses of their counterparts in the blowfly Calliphora, although we found substantial differences with respect to their dendritic structure and connectivity. This long-awaited functional characterization of HS cells in Drosophila provides the basis for the future dissection of optomotor behavior and the underlying neural circuitry by combining genetics, physiology, and behavior.

Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential for direct measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes (ΔF)in vivoand the relationship to the underlying neural activity and changes in intracellular calcium concentration (Δ[Ca2+]i) has not been given. We used two-photon microscopy, microinjection of synthetic Ca2+dyes andin vivocalibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca2+]iat rest and Δ[Ca2+]iat different action potential frequencies in presynaptic motoneuron boutons of transgenicDrosophilalarvae. We calibrated ΔFof eight different GECIsin vivoto neural activity, Δ[Ca2+]i, and ΔFof purified GECI protein at similar Δ[Ca2+]in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum ΔFcompared with YC3.3 and TN-L15in vivo. Maximum ΔFof GCaMP2 and GCaMP1.6 were almost identical. Small Δ[Ca2+]iwere reported best by YC3.60, D3cpv, and YC2.60. The kinetics of Δ[Ca2+]iwas massively distorted by all GECIs, with YC2.60 showing the slowest kinetics, whereas TN-XL exhibited the fastest decay. Single spikes were only reported by OGB-1; all GECIs were blind for Δ[Ca2+]iassociated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets.In vivo, theKD(dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum ΔFwas reduced. The latter could be attributed to resting [Ca2+]iand the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.

The genetic analysis of larval neuromuscular junctions (NMJs) ofDrosophilahas provided detailed insights into molecular mechanisms that control the morphological and physiological development of these glutamatergic synapses. However, because of the chronic defects caused by mutations, a time-resolved analysis of these mechanisms and their functional relationships has been difficult so far. In this study we provide a first temporal map of some of the molecular and cellular key processes, which are triggered in wild-type animals by natural larval locomotor activity and then mediate experience-dependent strengthening of larval NMJs. Larval locomotor activity was increased either by chronically rearing a larval culture at 29°C instead of 18 or 25°C or by acutely transferring larvae from a culture vial onto agar plates. Within 2 hr of enhanced locomotor activity, NMJs showed a significant potentiation of signal transmission that was rapidly reversed by an induced paralysis of the temperature-sensitive mutantparats1. Enhanced locomotor activity was also associated with a significant increase in the number of large subsynaptic translation aggregates. After 4 hr, postsynaptic DGluR-IIA glutamate receptor subunits started to transiently accumulate in ring-shaped areas around synapses, and they condensed later on, after chronic locomotor stimulation at 29°C, into typical postsynaptic patches. These NMJs showed a reduced perisynaptic expression of the cell adhesion molecule Fasciclin II, an increased number of junctional boutons, and significantly more active zones. Such temporal mapping of experience-dependent adaptations at developing wild-type and mutant NMJs will provide detailed insights into the dynamic control of glutamatergic signal transmission.

Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400% in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far.