Abstract: 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.
Abstract: Fly motion vision and resultant compensatory optomotor responses are a classic example for neural computation. Here we review our current understanding of processing of optic flow as generated by an animal's self-motion. Optic flow processing is accomplished in a series of steps: First, the time-varying photoreceptor signals are fed into a two-dimensional array of Reichardt-type elementary motion detectors (EMDs). EMDs compute, in parallel, local motion vectors at each sampling point in space. Second, the output signals of many EMDs are spatially integrated on the dendrites of large-field tangential cells in the lobula plate. In the third step, tangential cells form extensive interactions with each other, giving rise to their large and complex receptive fields. Thus, tangential cells can act as matched filters tuned to optic flow during particular flight maneuvers. They finally distribute their information onto postsynaptic descending neurons, which either instruct the motor centers of the thoracic ganglion for flight and locomotion control or act themselves as motor neurons that control neck muscles for head movements.
Abstract: 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.
Abstract: 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.
Abstract: The crystalline-like structure of the optic lobes of the fruit fly Drosophila melanogaster has made them a model system for the study of neuronal cell-fate determination, axonal path finding, and target selection. For functional studies, however, the small size of the constituting visual interneurons has so far presented a formidable barrier. We have overcome this problem by establishing in vivo whole-cell recordings from genetically targeted visual interneurons of Drosophila. Here, we describe the response properties of six motion-sensitive large-field neurons in the lobula plate that form a network consisting of individually identifiable, directionally selective cells most sensitive to vertical image motion (VS cells). Individual VS cell responses to visual motion stimuli exhibit all the characteristics that are indicative of presynaptic input from elementary motion detectors of the correlation type. Different VS cells possess distinct receptive fields that are arranged sequentially along the eye's azimuth, corresponding to their characteristic cellular morphology and position within the retinotopically organized lobula plate. In addition, lateral connections between individual VS cells cause strongly overlapping receptive fields that are wider than expected from their dendritic input. Our results suggest that motion vision in different dipteran fly species is accomplished in similar circuitries and according to common algorithmic rules. The underlying neural mechanisms of population coding within the VS cell network and of elementary motion detection, respectively, can now be analyzed by the combination of electrophysiology and genetic intervention in Drosophila.
Abstract: 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 (DeltaF) in vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration (Delta[Ca(2+)](i)) has not been given. We used two-photon microscopy, microinjection of synthetic Ca(2+) dyes and in vivo calibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca(2+)](i) at rest and Delta[Ca(2+)](i) at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated DeltaF of eight different GECIs in vivo to neural activity, Delta[Ca(2+)](i), and DeltaF of purified GECI protein at similar Delta[Ca(2+)] in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum DeltaF compared with YC3.3 and TN-L15 in vivo. Maximum DeltaF of GCaMP2 and GCaMP1.6 were almost identical. Small Delta[Ca(2+)](i) were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of Delta[Ca(2+)](i) was 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 Delta[Ca(2+)](i) associated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets. In vivo, the K(D) (dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum DeltaF was reduced. The latter could be attributed to resting [Ca(2+)](i) and the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.
Abstract: Neurons in the nervous system can change their functional properties over time. At present, there are no techniques that allow reliable monitoring of changes within identified neurons over repeated experimental sessions. We increased the signal strength of troponin C-based calcium biosensors in the low-calcium regime by mutagenesis and domain rearrangement within the troponin C calcium binding moiety to generate the indicator TN-XXL. Using in vivo two-photon ratiometric imaging, we show that TN-XXL exhibits enhanced fluorescence changes in neurons of flies and mice. TN-XXL could be used to obtain tuning curves of orientation-selective neurons in mouse visual cortex measured repeatedly over days and weeks. Thus, the genetically encoded calcium indicator TN-XXL allows repeated imaging of response properties from individual, identified neurons in vivo, which will be crucial for gaining new insights into cellular mechanisms of plasticity, regeneration and disease.
Abstract: In flies, the large tangential cells of the lobula plate represent an important processing center for visual navigation based on optic flow. Although the visual response properties of these cells have been well studied in blowflies, information on their synaptic organization is mostly lacking. Here we study the distribution of presynaptic release and postsynaptic inhibitory sites in the same set of cells in Drosophila melanogaster. By making use of transgenic tools and immunohistochemistry, our results suggest that HS and VS cells of Drosophila express gamma-aminobutyric acid (GABA) receptors in their dendritic region within the lobula plate, thus being postsynaptic to inhibitory input there. At their axon terminals in the protocerebrum, both cell types express synaptobrevin, suggesting the presence of presynaptic specializations there. HS- and VS-cell terminals additionally show evidence for postsynaptic GABAergic input, superimposed on this synaptic polarity. Our findings are in line with the general circuit for visual motion detection and receptive field properties as postulated from electrophysiological and optical recordings in blowflies, suggesting a similar functional organization of lobula plate tangential cells in the two species.
Abstract: 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.
Abstract: 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.
Abstract: Genetically encoded fluorescent probes of neural activity represent new promising tools for systems neuroscience. Here, we present a comparative in vivo analysis of 10 different genetically encoded calcium indicators, as well as the pH-sensitive synapto-pHluorin. We analyzed their fluorescence changes in presynaptic boutons of the Drosophila larval 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 for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of the optical signals.
Abstract: The genetic analysis of larval neuromuscular junctions (NMJs) of Drosophila has 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 degrees C instead of 18 or 25 degrees 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 mutant parats1. 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 degrees 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.
Abstract: 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.
Abstract: In this study we established a transgenic Ca2+ imaging technique in Drosophila that enabled us to target the Ca2+ sensor protein yellow Cameleon-2 specifically to larval neurons. This noninvasive method allowed us to measure evoked Ca2+ signals in presynaptic terminals of larval neuromuscular junctions (NMJs). We combined transgenic Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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.
Abstract: Long-term synaptic plasticity may be associated with structural rearrangements within the neuronal circuitry. Although the molecular mechanisms governing such activity-controlled morphological alterations are mostly elusive, polysomal accumulations at the base of developing dendritic spines and the activity-induced synthesis of synaptic components suggest that localized translation is involved during synaptic plasticity. Here we show that large aggregates of translational components as well as messenger RNA of the postsynaptic glutamate receptor subunit DGluR-IIA are localized within subsynaptic compartments of larval neuromuscular junctions of Drosophila melanogaster. Genetic models of junctional plasticity and genetic manipulations using the translation initiation factors eIF4E and poly(A)-binding protein showed an increased occurrence of subsynaptic translation aggregates. This was associated with a significant increase in the postsynaptic DGluR-IIA protein levels and a reduction in the junctional expression of the cell-adhesion molecule Fasciclin II. In addition, the efficacy of junctional neurotransmission and the size of larval neuromuscular junctions were significantly increased. Our results therefore provide evidence for a postsynaptic translational control of long-term junctional plasticity.
Abstract: In view of the prominent role of voltage-activated conductances in both neuronal differentiation and signal transmission, the present study describes developmental alterations of ion channel properties during the functional maturation of rat RGCs, i.e., between embryonic day 15 when RGCs start to differentiate (E15) and postnatal day 35 (P35) when the retina is fully developed and the animals had already visual input for about 2 weeks. While the sodium system seems to reach maturity already at the end of the second postnatal week, significant alterations in the potassium system were found only from postnatal day 10 on. The functional implications of these alterations are discussed.
Abstract: Ca2(+)-independent voltage-activated potassium currents were investigated during the differentiation of rat retinal ganglion cells. Whole-cell patch-clamp recordings of Ca2(+)-independent voltage-activated potassium currents and their individual current components, i.e. a sustained, tetraethylammonium-sensitive current, a transient, 4-aminopyridine-sensitive current, and a slowly decaying current that was blocked by Ba2+, revealed distinct ontogenetic modifications in current densities and in activation and inactivation parameters. All three current types were expressed simultaneously at embryonic day 17/18 and were present in all retinal ganglion cells thereafter without showing any significant changes until the end of the first postnatal week. Ca2(+)-independent voltage-activated potassium current densities then increased strongly from postnatal day 8 onwards. Tetraethylammonium-sensitive current density increased about eightfold from 74 pA/pF in embryonic stages to 586 pA/pF in adult cells, whereas the transient potassium currents blocked by 4-aminopyridine increased only about 2.5-fold from 174 pA/pF to 442 pA/pF. The Ba2(+)-sensitive current increased simultaneously from 35 pA/pF to 332 pA/pF. The much higher increase in the sustained current components during retinal ganglion cell differentiation accounted for the changes in decay kinetics of Ca2(+)-independent voltage-activated potassium current observed in later postnatal stages. Alterations in current densities were paralleled by pronounced changes in current kinetics. From postnatal day 8 onwards, activation of Ca2(+)-independent voltage-activated potassium current was right-shifted for about 10 mV owing to a shift in tetraethylammonium-sensitive current-activation, whereas activation of other K+ components remained unaltered. Tetraethylammonium-sensitive current steady-state inactivation was incomplete at all developmental stages. About 50% of the tetraethylammonium-sensitive current elicited by a depolarization to +36 mV did not inactivate after prepulse potentials positive to -10 mV. In contrast, transient potassium current blocked by 4-aminopyridine almost fully inactivated during embryonic stages, whereas in adult retinal ganglion cells about 40% of this current component did not inactivate after prepulse potentials positive to -20 mV. Parallel investigation of the resting membrane potential during retinal ganglion cells differentiation showed an exponential increase from -3 mV at embryonic day 15/16 when no voltage-activated ion currents were expressed to a final value of -58 mV at postnatal day 8. These results show that fundamental potassium current modifications occur relatively late in retinal ganglion cell development and only after the resting potential is at its final value.
