Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron.

Spatial readout of visual looming in the central brain of .

Visual systems can exploit spatial correlations in the visual scene by using retinotopy, the organizing principle by which neighboring cells encode neighboring spatial locations. However, retinotopy is often lost, such as when visual pathways are integrated with other sensory modalities. How is spatial information processed outside of strictly visual brain areas? Here, we focused on visual looming responsive LC6 cells in , a population whose dendrites collectively cover the visual field, but whose axons form a single glomerulus-a structure without obvious retinotopic organization-in the central brain.

A general approach to engineer positive-going eFRET voltage indicators.

Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity.

jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range.

A Neural Circuit Encoding the Experience of Copulation in Female Drosophila.

Female behavior changes profoundly after mating. In Drosophila, the mechanisms underlying the long-term changes led by seminal products have been extensively studied. However, the effect of the sensory component of copulation on the female's internal state and behavior remains elusive.

Neuronal reactivation during post-learning sleep consolidates long-term memory in .

Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood.

Mapping Neurotransmitter Identity in the Whole-Mount Brain Using Multiplex High-Throughput Fluorescence Hybridization.

Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence hybridization (FISH) enables direct labeling of type-specific mRNA in neurons.

Communication from Learned to Innate Olfactory Processing Centers Is Required for Memory Retrieval in Drosophila.

The behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly.

Inverse-response Ca indicators for optogenetic visualization of neuronal inhibition.

We have developed a series of yellow genetically encoded Ca indicators for optical imaging (Y-GECOs) with inverted responses to Ca and apparent dissociation constants (K') ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca indicators, we expressed the four highest affinity variants (K's = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca in layers 8-10, and a corresponding increase in Y-GECO fluorescence.

The functional organization of descending sensory-motor pathways in .

In most animals, the brain controls the body via a set of descending neurons (DNs) that traverse the neck. DN activity activates, maintains or modulates locomotion and other behaviors. Individual DNs have been well-studied in species from insects to primates, but little is known about overall connectivity patterns across the DN population.

Behavioral state modulates the ON visual motion pathway of .

The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood.

A Circuit Node that Integrates Convergent Input from Neuromodulatory and Social Behavior-Promoting Neurons to Control Aggression in Drosophila.

Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. We have performed a systematic, unbiased screen to identify OA receptor-expressing neurons (OARNs) that control aggression in Drosophila.

A connectome of a learning and memory center in the adult brain.

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In , the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB's α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy.

Quantitative mRNA imaging throughout the entire Drosophila brain.

We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).

The Emergence of Directional Selectivity in the Visual Motion Pathway of Drosophila.

The perception of visual motion is critical for animal navigation, and flies are a prominent model system for exploring this neural computation. In Drosophila, the T4 cells of the medulla are directionally selective and necessary for ON motion behavioral responses. To examine the emergence of directional selectivity, we developed genetic driver lines for the neuron types with the most synapses onto T4 cells.

Doublesex Regulates the Connectivity of a Neural Circuit Controlling Drosophila Male Courtship Song.

It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The Drosophila melanogaster male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively.

Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship.

Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities.

Calcium imaging in the Drosophila olfactory system with a genetic indicator.

Insects show sophisticated odor-mediated behaviors controlled by an olfactory system that is genetically and anatomically simpler than that of vertebrates, providing an attractive system to investigate the mechanistic link between behavior and odor perception. Advances in neuroscience have been facilitated by modern optical imaging technologies--both in instrumentation and in probe design--that permit the visualization of functional neural circuits. Imaging calcium activity in genetically defined populations of neurons provides an important tool for investigating the function of neural circuits.

Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo.

Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin in vivo have not been reported.

Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing.

Behavior cannot be predicted from a "connectome" because the brain contains a chemical "map" of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. Here we describe a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain.

Beat-frequency adjustable Er3+-doped DBR fiber laser for ultrasound detection.

A compact low beat-frequency dual-polarization distributed Bragg reflector (DBR) fiber laser whose beat frequency can be varied, for high-frequency ultrasound detection has been proposed and experimentally demonstrated. The laser was fabricated in small birefringent commercial erbium-doped fiber. It operated in a robust single-longitude mode with output power of more than 1 mW and high signal-to-noise ratio better than 60 dB.

Single tilted Bragg reflector fiber laser for simultaneous sensing of refractive index and temperature.

A type of fiber laser, called tilted Bragg reflector fiber laser (TBR-FL), is proposed and its application in simultaneous sensing of surrounding refractive index (SRI) and temperature is demonstrated. This FL is formed by a pair of wavelength and tilt-angle matched tilted fiber Bragg gratings (TFBGs) that acted both as a resonant cavity and sensing element. A unique spectral feature of the TBR-FL is the presence of grating tilt-induced cladding modes spectrum that does not appear in other type of FL, which provides an extra sensing mechanism.

Fourier analysis for hydrostatic pressure sensing in a polarization-maintaining photonic crystal fiber.

We measured the hydrostatic pressure dependence of the birefringence and birefringent dispersion of a Sagnac interferometric sensor incorporating a length of highly birefringent photonic crystal fiber using Fourier analysis. Sensitivity of both the phase and chirp spectra to hydrostatic pressure is demonstrated. Using this analysis, phase-based measurements showed a good linearity with an effective sensitivity of 9.

Highly sensitive bending sensor based on Er3+-doped DBR fiber laser.

A short cavity Er(3+)-doped distributed-Bragg-reflector (DBR) fiber laser with a low polarization beat frequency has been demonstrated for bending measurement. The polarization beat frequency of the DBR laser is extremely sensitive to bending and can measure curvature changes as small as 1.8 x 10(-2) m(-1).