NOSA, an Analytical Toolbox for Multicellular Optical Electrophysiology.

Understanding how neural networks generate activity patterns and communicate with each other requires monitoring the electrical activity from many neurons simultaneously. Perfectly suited tools for addressing this challenge are genetically encoded voltage indicators (GEVIs) because they can be targeted to specific cell types and optically report the electrical activity of individual, or populations of neurons. However, analyzing and interpreting the data from voltage imaging experiments is challenging because high recording speeds and properties of current GEVIs yield only low signal-to-noise ratios, making it necessary to apply specific analytical tools. Here, we present NOSA (Neuro-Optical Signal Analysis), a novel open source software designed for analyzing voltage imaging data and identifying temporal interactions between electrical activity patterns of different origin. In this work, we explain the challenges that arise during voltage imaging experiments and provide hands-on analytical solutions. We demonstrate how NOSA's baseline fitting, filtering algorithms and movement correction can compensate for shifts in baseline fluorescence and extract electrical patterns from low signal-to-noise recordings. NOSA allows to efficiently identify oscillatory frequencies in electrical patterns, quantify neuronal response parameters and moreover provides an option for analyzing simultaneously recorded optical and electrical data derived from patch-clamp or other electrode-based recordings. To identify temporal relations between electrical activity patterns we implemented different options to perform cross correlation analysis, demonstrating their utility during voltage imaging in Drosophila and mice. All features combined, NOSA will facilitate the first steps into using GEVIs and help to realize their full potential for revealing cell-type specific connectivity and functional interactions.

Network-Specific Synchronization of Electrical Slow-Wave Oscillations Regulates Sleep Drive in Drosophila.

Slow-wave rhythms characteristic of deep sleep oscillate in the delta band (0.5-4 Hz) and can be found across various brain regions in vertebrates. Across phyla, however, an understanding of the mechanisms underlying oscillations and how these link to behavior remains limited. Here, we discover compound delta oscillations in the sleep-regulating R5 network of Drosophila. We find that the power of these slow-wave oscillations increases with sleep need and is subject to diurnal variation. Optical multi-unit voltage recordings reveal that single R5 neurons get synchronized by activating circadian input pathways. We show that this synchronization depends on NMDA receptor (NMDAR) coincidence detector function, and that an interplay of cholinergic and glutamatergic inputs regulates oscillatory frequency. Genetically targeting the coincidence detector function of NMDARs in R5, and thus the uncovered mechanism underlying synchronization, abolished network-specific compound slow-wave oscillations. It also disrupted sleep and facilitated light-induced wakening, establishing a role for slow-wave oscillations in regulating sleep and sensory gating. We therefore propose that the synchronization-based increase in oscillatory power likely represents an evolutionarily conserved, potentially "optimal," strategy for constructing sleep-regulating sensory gates.

A new method to characterize function of the Drosophila heart by means of optical flow.

The minuteness of Drosophila poses a challenge to quantify performance of its tubular heart and computer-aided analysis of its beating heart has evolved as a resilient compromise between instrumental costs and data robustness. Here, we introduce an optical flow algorithm (OFA) that continuously registers coherent movement within videos of the beating Drosophila heart and uses this information to subscribe the time course of observation with characteristic phases of cardiac contraction or relaxation. We report that the OFA combines high discriminatory power with robustness to characterize the performance of the Drosophila tubular heart using indicators from human cardiology. We provide proof of this concept using the test bed of established cardiac conditions that include the effects of ageing, knockdown of the slow repolarizing potassium channel subunit KCNQ and ras-mediated hypertrophy of the heart tube. Together, this establishes the analysis of coherent movement as a suitable indicator of qualitative changes of the heart's beating characteristics, which improves the usefulness of Drosophila as a model of cardiac diseases.

Goggatomy: A Method for Opening Small Cuticular Compartments in Arthropods for Physiological Experiments.

Most sense organs of arthropods are ensconced in small exoskeletal compartments that hinder direct access to plasma membranes. We have developed a method for exposing live sensory and supporting cells in such structures. The technique uses a viscous light cured resin to embed and support the structure, which is then sliced with a sharp blade. We term the procedure a "goggatomy," from the Khoisan word for a bug, gogga. To demonstrate the utility of the method we show that it can be used to expose the auditory chordotonal organs in the second antennal segment and the olfactory receptor neurons in the third antennal segment of Drosophila melanogaster, preserving the transduction machinery. The procedure can also be used on other small arthropods, like mosquitoes and mites to expose a variety of cells.

Presynaptic GABA Receptors Mediate Temporal Contrast Enhancement in Drosophila Olfactory Sensory Neurons and Modulate Odor-Driven Behavioral Kinetics.

Contrast enhancement mediated by lateral inhibition within the nervous system enhances the detection of salient features of visual and auditory stimuli, such as spatial and temporal edges. However, it remains unclear how mechanisms for temporal contrast enhancement in the olfactory system can enhance the detection of odor plume edges during navigation. To address this question, we delivered to Drosophila melanogaster flies pulses of high odor intensity that induce sustained peripheral responses in olfactory sensory neurons (OSNs). We use optical electrophysiology to directly measure electrical responses in presynaptic terminals and demonstrate that sustained peripheral responses are temporally sharpened by the combined activity of two types of inhibitory GABA receptors to generate contrast-enhanced voltage responses in central OSN axon terminals. Furthermore, we show how these GABA receptors modulate the time course of innate behavioral responses after odor pulse termination, demonstrating an important role for temporal contrast enhancement in odor-guided navigation.

Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila.

BACKGROUND: Imbalances in amount and timing of sleep are harmful to physical and mental health. Therefore, the study of the underlying mechanisms is of great biological importance. Proper timing and amount of sleep are regulated by both the circadian clock and homeostatic sleep drive. However, very little is known about the cellular and molecular mechanisms by which the circadian clock regulates sleep. In this study, we describe a novel role for diuretic hormone 31 (DH31), the fly homolog of the vertebrate neuropeptide calcitonin gene-related peptide, as a circadian wake-promoting signal that awakens the fly in anticipation of dawn. RESULTS: Analysis of loss-of-function and gain-of-function Drosophila mutants demonstrates that DH31 suppresses sleep late at night. DH31 is expressed by a subset of dorsal circadian clock neurons that also express the receptor for the circadian neuropeptide pigment-dispersing factor (PDF). PDF secreted by the ventral pacemaker subset of circadian clock neurons acts on PDF receptors in the DH31-expressing dorsal clock neurons to increase DH31 secretion before dawn. Activation of PDF receptors in DH31-positive DN1 specifically affects sleep and has no effect on circadian rhythms, thus constituting a dedicated locus for circadian regulation of sleep. CONCLUSIONS: We identified a novel signaling molecule (DH31) as part of a neuropeptide relay mechanism for circadian control of sleep. Our results indicate that outputs of the clock controlling sleep and locomotor rhythms are mediated via distinct neuronal pathways.

Temporal integration of cholinergic and GABAergic inputs in isolated insect mushroom body neurons exposes pairing-specific signal processing.

GABAergic modulation of neuronal activity plays a crucial role in physiological processes including learning and memory in both insects and mammals. During olfactory learning in honeybees (Apis mellifera) and Drosophila melanogaster the temporal relation between excitatory cholinergic and inhibitory GABAergic inputs critically affects learning. However, the cellular mechanisms of temporal integration of these antagonistic inputs are unknown. To address this question, we use calcium imaging of isolated honeybee and Drosophila Kenyon cells (KCs), which are targets of cholinergic and GABAergic inputs during olfactory learning. In the whole population of honeybee KCs we find that pairing of acetylcholine (ACh) and γ-aminobutyric acid (GABA) Comment: Please use the greek letter for gamma reduces the ACh-induced calcium influx, and depending on their temporal sequence, induces different forms of neuronal plasticity. After ACh-GABA pairing the calcium influx of a subsequent excitatory stimulus is increased, while GABA-ACh pairing affects the decay time leading to elevated calcium levels during the late phase of a subsequent excitatory stimulus. In an exactly defined subset of Drosophila KCs implicated in learning we find similar pairing-specific differences. Specifically the GABA-ACh pairing splits the KCs in two functional subgroups: one is only weakly inhibited by GABA and shows no neuronal plasticity and the other subgroup is strongly inhibited by GABA and shows elevated calcium levels during the late phase of a subsequent excitatory stimulus. Our findings provide evidence that insect KCs are capable of contributing to temporal processing of cholinergic and GABAergic inputs, which provides a neuronal mechanism of the differential temporal role of GABAergic inhibition during learning.

Genetically targeted optical electrophysiology in intact neural circuits.

Nervous systems process information by integrating the electrical activity of neurons in complex networks. This motivates the long-standing interest in using optical methods to simultaneously monitor the membrane potential of multiple genetically targeted neurons via expression of genetically encoded fluorescent voltage indicators (GEVIs) in intact neural circuits. No currently available GEVIs have demonstrated robust signals in intact brain tissue that enable reliable recording of individual electrical events simultaneously in multiple neurons. Here, we show that the recently developed "ArcLight" GEVI robustly reports both subthreshold events and action potentials in genetically targeted neurons in the intact Drosophila fruit fly brain and reveals electrical signals in neurite branches. In the same way that genetically encoded fluorescent sensors have revolutionized the study of intracellular Ca(2+) signals, ArcLight now enables optical measurement in intact neural circuits of membrane potential, the key cellular parameter that underlies neuronal information processing.

Focal uncaging of GABA reveals a temporally defined role for GABAergic inhibition during appetitive associative olfactory conditioning in honeybees.

Throughout the animal kingdom, the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is a key modulator of physiological processes including learning. With respect to associative learning, the exact time in which GABA interferes with the molecular events of learning has not yet been clearly defined. To address this issue, we used two different approaches to activate GABA receptors during appetitive olfactory conditioning in the honeybee. Injection of GABA-A receptor agonist muscimol 20 min before but not 20 min after associative conditioning affects memory performance. These memory deficits were attenuated by additional training sessions. Muscimol has no effect on sensory perception, odor generalization, and nonassociative learning, indicating a specific role of GABA during associative conditioning. We used photolytic uncaging of GABA to identify the GABA-sensitive time window during the short pairing of the conditioned stimulus (CS) and the unconditioned stimulus (US) that lasts only seconds. Either uncaging of GABA in the antennal lobes or the mushroom bodies during the CS presentation of the CS-US pairing impairs memory formation, while uncaging GABA during the US phase has no effect on memory. Uncaging GABA during the CS presentation in memory retrieval also has no effect. Thus, in honeybee appetitive olfactory learning GABA specifically interferes with the integration of CS and US during associative conditioning and exerts a modulatory role in memory formation depending on the training strength.