Social isolation strongly modulates behavior across the animal kingdom. We utilized the fruit fly
to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq ...identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors.
knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of
neurons each similarly increased isolation-driven aggression. Our results suggest a U-shaped dependence of social isolation-induced aggressive behavior on
signaling, similar to the actions of many neuromodulators in other contexts.
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The ...state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons ...in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.
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.
Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in ...this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the ∼10-nm localization precision of its parent.
Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high ...concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.
We developed a multicolor neuron labeling technique in Drosophila melanogaster that combines the power to specifically target different neural populations with the label diversity provided by ...stochastic color choice. This adaptation of vertebrate Brainbow uses recombination to select one of three epitope-tagged proteins detectable by immunofluorescence. Two copies of this construct yield six bright, separable colors. We used Drosophila Brainbow to study the innervation patterns of multiple antennal lobe projection neuron lineages in the same preparation and to observe the relative trajectories of individual aminergic neurons. Nerve bundles, and even individual neurites hundreds of micrometers long, can be followed with definitive color labeling. We traced motor neurons in the subesophageal ganglion and correlated them to neuromuscular junctions to identify their specific proboscis muscle targets. The ability to independently visualize multiple lineage or neuron projections in the same preparation greatly advances the goal of mapping how neurons connect into circuits.
Multiphoton imaging (MPI) is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. We combined regenerative amplification multiphoton microscopy ...(RAMM) with genetically encoded calcium indicators to extend MPI of neuronal population activity into layer 5 (L5) of adult mouse somatosensory cortex. We found that this approach could be used to record and quantify spontaneous and sensory-evoked activity in populations of L5 neuronal somata located as much as 800 μm below the pia. In addition, we found that RAMM could be used to simultaneously image activity from large (80) populations of apical dendrites and follow these dendrites down to their somata of origin.
Alpha/Y-type retinal ganglion cells encode visual information with a receptive field composed of nonlinear subunits. This nonlinear subunit structure enhances sensitivity to patterns composed of high ...spatial frequencies. The Y-cell's subunits are the presynaptic bipolar cells, but the mechanism for the nonlinearity remains incompletely understood. We investigated the synaptic basis of the subunit nonlinearity by combining whole-cell recording of mouse Y-type ganglion cells with two-photon fluorescence imaging of a glutamate sensor (iGluSnFR) expressed on their dendrites and throughout the inner plexiform layer. A control experiment designed to assess iGluSnFR's dynamic range showed that fluorescence responses from Y-cell dendrites increased proportionally with simultaneously recorded excitatory current. Spatial resolution was sufficient to readily resolve independent release at intermingled ON and OFF bipolar terminals. iGluSnFR responses at Y-cell dendrites showed strong surround inhibition, reflecting receptive field properties of presynaptic release sites. Responses to spatial patterns located the origin of the Y-cell nonlinearity to the bipolar cell output, after the stage of spatial integration. The underlying mechanism differed between OFF and ON pathways: OFF synapses showed transient release and strong rectification, whereas ON synapses showed relatively sustained release and weak rectification. At ON synapses, the combination of fast release onset with slower release offset explained the nonlinear response of the postsynaptic ganglion cell. Imaging throughout the inner plexiform layer, we found transient, rectified release at the central-most levels, with increasingly sustained release near the borders. By visualizing glutamate release in real time, iGluSnFR provides a powerful tool for characterizing glutamate synapses in intact neural circuits.
Although most proteins conform to the classical one‐structure/one‐function paradigm, an increasing number of proteins with dual structures and functions have been discovered. In response to cellular ...stimuli, such proteins undergo structural changes sufficiently dramatic to remodel even their secondary structures and domain organization. This “fold‐switching” capability fosters protein multi‐functionality, enabling cells to establish tight control over various biochemical processes. Accurate predictions of fold‐switching proteins could both suggest underlying mechanisms for uncharacterized biological processes and reveal potential drug targets. Recently, we developed a prediction method for fold‐switching proteins using structure‐based thermodynamic calculations and discrepancies between predicted and experimentally determined protein secondary structure (Porter and Looger, Proc Natl Acad Sci U S A 2018; 115:5968–5973). Here we seek to leverage the negative information found in these secondary structure prediction discrepancies. To do this, we quantified secondary structure prediction accuracies of 192 known fold‐switching regions (FSRs) within solved protein structures found in the Protein Data Bank (PDB). We find that the secondary structure prediction accuracies for these FSRs vary widely. Inaccurate secondary structure predictions are strongly associated with fold‐switching proteins compared to equally long segments of non‐fold‐switching proteins selected at random. These inaccurate predictions are enriched in helix‐to‐strand and strand‐to‐coil discrepancies. Finally, we find that most proteins with inaccurate secondary structure predictions are underrepresented in the PDB compared with their alternatively folded cognates, suggesting that unequal representation of fold‐switching conformers within the PDB could be an important cause of inaccurate secondary structure predictions. These results demonstrate that inconsistent secondary structure predictions can serve as a useful preliminary marker of fold switching.
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