ABSTRACTS OF PAPERS AT THE SEVENTY-FIRST ANNUAL MEETING OF THE SOCIETY OF GENERAL PHYSIOLOGISTS: The Optical Revolution in Physiology: From Membrane to Brain

1. All-Optical Electrophysiology in Behaving Mice With Enhanced Near Infrared Voltage Sensors. YOAV ADAM,¹ SHAN LOU,¹ and ADAM COHEN,^{1,2 1}Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA; ²Howard Hughes Medical Institute

The ability to record and perturb optically from multiple neurons in the intact brain at single spike resolution will open new frontiers in neuroscience research. We combined protein engineering, novel microscopy techniques and targeted gene expression to develop a set of tools for “all-optical electrophysiology” in the brain of awake, behaving mice. We first engineered an improved near infrared voltage sensor called QuasAr3, which allowed high-fidelity recording of somatic and dendritic action potentials and sub-threshold events in acute brain slices. We then developed a Cre-dependent transgenic mouse line expressing both QuasAr3 and the optically orthogonal channelrhodopsin CheRiff and used it for all-optical classification of neuronal subtypes based on their firing patterns. We also developed an indicator called paQuasAr3, a photo activated variant that showed brighter fluorescence and improved signal-to-noise ratio (SNR) upon activation with blue light. Sparse expression of paQuasAr3 reported spontaneous spiking activity at high SNR in the hippocampus (CA1) and olfactory bulb of anesthetized mice.

We next sought to record from and manipulate multiple cells simultaneously. We developed soma-restricted versions of QuasAr3, paQuasAr3, and CheRiff, which enabled dense expression by significantly reducing the neuropil background. To further improve the imaging contrast, reduce the overall laser power, and allow precise spatial control, we patterned the stimulation and imaging lasers with a digital micromirror device. The combination of dense expression and patterned illumination allowed us to record from multiple cells, simultaneously in the hippocampal CA1 region of behaving mice and to detect running dependent supra- and subthreshold voltage dynamics. This set of tools opens the possibility to decipher neuronal circuits at high spatiotemporal resolution and unprecedented throughput.

2. Neuronal Responses of Ca²⁺ Transients in Interstitial Cells of Cajal in the Small Bowel. SALAH A. BAKER, BERNARD T. DRUMM and KENTON M. SANDERS, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557

Introduction: Interstitial cells of Cajal (ICC) regulate of smooth muscle excitability and motility in the gastrointestinal (GI) tract. ICC in the deep muscular plexus (ICC-DMP) of the small intestine are closely associated with varicosities of enteric motor neurons and are suggested to transduce neural responses. ICC-DMP fire spontaneous Ca²⁺ transients linked to activation of Ca²⁺-activated Cl⁻ channels, but modulation of Ca²⁺ transients by enteric neural inputs has not been described in detail.

Aim: Measure Ca²⁺ transients in ICC-DMP in situ that are elicited or suppressed by excitatory and inhibitory motor neural inputs.

Methods: We used the Cre-loxP technique to express GCaMP3 in combination with confocal imaging. Electrical field stimulation (EFS) was used to activate enteric motor neurons.

Results: EFS elicited inhibitory and excitatory effects on Ca²⁺ transients. Inhibitory nerve stimulation suppressed Ca²⁺ transients in ICC-DMP (from 66 transients/min to 2.9 transients/min). At termination of EFS, there was a period (∼5–12s) of greatly enhanced Ca²⁺ transient activity (increased to 125 transients/min). Inhibitory effects on Ca²⁺ transients were mediated mainly by nitrergic neurotransmission, as the NO synthase inhibitor L-NNA blocked inhibitory effects. Prior to EFS, L-NNA increased spontaneous Ca²⁺ transients, and the NO donor, NONATE, inhibited Ca²⁺ transients. Furthermore, the soluble guanylate cyclase activator, BAY 58-2667, also decreased Ca²⁺ transients in ICC, indicating the inhibitory effects of nitrergic neurotransmission in ICC-DMP are mediated by the GC-cGMP pathway. Excitatory Ca²⁺ responses in ICC were reduced by atropine and the neurokinin 1 receptor (NK1) antagonist RP-67580 but not by an NK2 antagonist, MEN-10376. Exogenous ACh and substance P increased Ca²⁺ transients while atropine and RP-67580 decreased Ca²⁺ transients in ICC-DMP.

Conclusions: Excitatory and inhibitory neurotransmitters modulate Ca²⁺ transients in ICC-DMP. These observations demonstrate direct innervation and post-junctional responsiveness of ICC-DMP in intestinal muscles. Ca²⁺ transients in ICC-DMP activate inward currents that can regulate the excitability of the small intestine provides a signaling mechanism responsible for transduction of enteric neuronal input.

3. Super-Resolution STORM Quantitative Analysis of Nanodomain Protein Interactions in Neurons. CHASE M. CARVER and MARK S. SHAPIRO, Department of Cellular and Integrative Physiology, University of Texas Health Science Center San Antonio, TX

Fidelity of neuronal signaling requires organization of signaling molecules into highly proximal macromolecular complexes. The intrinsic diffraction limit of light makes visualization of individual signaling complexes difficult. However, using multi-color super-resolution stochastic optical reconstruction microscopy (STORM), we observed intimate association of individual molecules within signaling complexes containing ion channels (M-type K⁺, L-type Ca²⁺, or TRPV1 channels) and G protein–coupled receptors coupled by A-kinase–anchoring protein (AKAP)79/150 as a scaffold. We identified novel interplay of complexes containing diverse channel types in sensory neurons, dependent on AKAP79/150. Electrophysiological studies confirmed functional coupling. We also determined the capability of STORM to image physical interactions spanning the ER and plasma membrane.

Beyond nearest-neighbor localization analysis, STORM-derived localizations can be algorithmically characterized by proximity and density to signify interactions of the labeled proteins. We developed an in-house density-based clustering (DBSCAN-like) algorithm to objectively categorize labeled protein interactions with stringent parameters, rather than selecting clusters by eye. We used population statistics of STORM cluster data derived from a large sample of cells to analyze cluster size, cluster localization number, and overall percentage of each possible cluster outcome derived from two- or three-color labeling. Therefore, we used the total population distributions of clusters categorized by type and size to probabilistically determine various states of channel interactions.

Our findings illustrate the novel role of AKAP150 as a molecular coupler of different channels with unique functions within single nanodomains in tuning the physiological response of sensory neurons. The cluster analysis demonstrates macromolecular complexes in competition for AKAP150, evident from the limited existence of unpaired AKAP150 in sensory neurons. We demonstrate a novel methodology for quantitatively understanding spatial interactions under STORM. Such methods can visualize dynamic states of channel–complex interactions that mirror structural and functional changes in neuronal signaling.

4. Optical Imaging and Labeling of Individual Biomolecules in Dense Clusters. MINGJIE DAI,^1,2,3 RALF JUNGMANN,^1,2,4 NINNING LIU,^1,2 and PENG YIN,^{1,2 1}Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115; ²Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115; ³Biophysics Program, Harvard University, Boston, Massachusetts 02115; ⁴Present address: Max Planck Institute of Biochemistry and LMU, Munich 82152, Germany

Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and synthetic nanostructures down to ∼15 nm in size to be imaged. However, direct optical observation of individual molecular targets (∼5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography)—a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridization—on synthetic DNA nanostructures. We examined the effects of high photon count, high blinking statistics, and appropriate blinking duty cycle on imaging quality and developed a software-based drift correction method that achieves <1-nm residual drift (r.m.s.) over hours. This allowed us to image a densely packed triangular lattice pattern with ∼5-nm point-to-point distance, and analyze DNA origami structural offset with angstrom-level precision (2 Å) from single-molecule studies. By combining the approach with multiplexed Exchange-PAINT imaging, we further demonstrated an optical nano-display with 5 × 5–nm pixel size and three distinct colors and with <1-nm cross-channel registration accuracy. Combined with photo-activated cross-linker, we further demonstrated selective labeling on single molecule targets with custom patterns and subdiffraction (∼30 nm) resolution. These methods open up possibilities for direct and quantitative optical observation and perturbation of individual biomolecular features in crowded environments.

5. Increase in Spontaneous Neuronal Activity Occurs During a Stage of Vascular Expansion in the Rat Auditory Brainstem During Postnatal Development. MARIANO DI GUILMI,^1,2 CHEIKH BABOU,¹ and ADRIÁN RODRÍGUEZ-CONTRERAS,^{1 1}The City University of New York, City College, Biology Department; ²Laboratorio de Fisiología y Genética de Audición, INGEBI, Argentina.

Neurovascular coupling reflects the metabolic and signaling relationship between vascular endothelial and neural cells. However, little is known about the developmental origins of neurovascular coupling. Before the onset of hearing around postnatal day 12 (P12), auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB) of rats fire spontaneous bursts of action potentials (Tritsch et al. 2010. Nature Neurosci. 13:1050–1052), cell proliferation increases, and astrocytes differentiate (Saliu et al. 2104. J. Comp. Neurol. 522:971–985), suggesting that developmental changes may occur in neurovascular coupling. In this study, developmental changes in vascular network structure and neuronal ensemble activity were examined in the MNTB of rat pups. Development of the vascular network was examined with isolectin-B4 and EdU histochemistry in 80-µm thick horizontal brainstem sections (n = 19 pups). In separate experiments, ensemble neuronal activity was measured with 16-channel multi-electrode arrays placed in the MNTB of isoflurane or ketamine anesthetized pups (n = 30 pups). Although vascular volume (in µm³) was constant between P0 and P5 (15,879 ± 1,147 and 16,654 ± 906, respectively), a marked increase was detected at P10 (30,915 ± 2,502; Student’s t test, P < 0.00001). EdU labeling confirmed cell proliferation of vascular and perivascular cells at P6 and P7. Electrophysiology recordings showed that multi-unit activity could be detected as early as P3, but with a noticeable increase at P6. Altogether, this data shows coordinated changes in vascular volume and neuronal activity during the prehearing stage of rat postnatal development.

6. Fluorescent False Neurotransmitter 270: New Tracer for Examining Norepinephrine Synaptic Function In Vivo. MATTHEW DUNN, DAVID SULZER, and DALIBOR SAMES, Departments of Chemistry, Neurology, Pharmacology, and Psychiatry, Columbia University, New York, NY

Norepinephrine is a monoamine neurotransmitter with a wide repertoire of physiological roles in the peripheral and central nervous systems, including regulation of stress responses, arousal, and cognition. Despite major strides in understanding the noradrenergic system, limited experimental means for examining neurotransmitter release at discrete presynaptic boutons have prevented studying the functional properties of individual noradrenergic synapses in the brain. To address this need, we expanded the fluorescent false neurotransmitters (FFNs) platform developed in our laboratories to this neurotransmitter system. We identified FFN270 as a novel fluorescent and pH-sensitive substrate of both the norepinephrine transporter and the vesicular monoamine transporter. As such, FFN270 traces norepinephrine transmission using the native protein machinery already specific to these neurons, providing a novel approach to optically study the regulation of neurotransmitter uptake, vesicular loading, and synaptic vesicle content release of norepinephrine presynaptic synapses. Using FFN270, we were able to access important physiological parameters, such as synaptic release probability of individual terminals, changes in vesicular pH, and neurotransmitter release events even in sparsely innervated cortical areas. Through specific optogenetic activation of the norepinephrine system, we observed heterogeneous release properties from individual norepinephrine release sites in the barrel cortex, identifying distinct low and high releasing populations. We also report the first measurement of monoamine neurotransmitter release with single synapse resolution in the living mouse, using both optogenetics and pharmacological activation with amphetamine. In conclusion, FFN270 provides a readout of synaptic activity within the norepinephrine system that is currently not possible with other tools in the field and can prove invaluable in studying synaptic function and regulation.

7. Phenotypic Assays of Synaptic Transmission Using All-Optical Electrophysiology. JOHN M. FERRANTE, FELIPE GERHARD, HANSINI UPADHYAY, GABRIEL BORJA, HIMALI SHROFF, STEVEN NAGLE, LUIS A. WILLIAMS, OWEN MCMANUS, KIT WERLEY, and GRAHAM T. DEMPSEY, Q-State Biosciences Inc., Cambridge, MA, 02139

A multitude of studies have implicated synaptic dysfunction in neurological diseases, including forms of epilepsy, Alzheimer’s disease, Parkinson’s disease, autism spectrum disorder (ASD), schizophrenia, and affective disorders. However, conventional methods of measuring synaptic activity are limiting: manual patch clamp has exquisite information but throughput is very low, while fluorescent Ca⁺⁺ assays are high-throughput but cannot resolve subthreshold postsynaptic potentials (PSPs). To address these shortcomings, we developed synaptic assays based on Optopatch, which is comprised of an engineered channelrhodopsin, CheRiff, to stimulate cells with blue light, and an engineered fluorescent protein, QuasAr, to record voltage with red light. Using Cre-dependent constructs, we express CheRiff and QuasAr in mutually exclusive subsets of cells, allowing us to stimulate pre-synaptic cells and record PSPs from their postsynaptic partners. Our imaging system allows the simultaneous recordings of PSPs from ∼20–50 individual neurons within a 0.5 × 4–mm FOV with 1-ms temporal resolution. Using combinations of synaptic blockers, we pharmacologically isolate GABAR/NMDAR/AMPAR signals and extract excitatory and inhibitory PSP properties, including amplitude, rise time, decay time, width, and area from tens of thousands of cells in a single day. In addition to high-throughput assays of PSPs, the platform can interrogate other critical aspects of synaptic function. We will discuss preliminary results, including (1) interrogation of network connectivity and microcircuits by targeted stimulation of individual cells with a digital mirror device (DMD); (2) resolving synaptic behaviors between different neuronal types using selective promoters (e.g., dlx1); (3) studying long- and short-term potentiation (LTP and STP); and (4) quantifying NMDAR and AMPAR PSPs in human induced pluripotent stem (hIPS) cell–derived excitatory neurons. The high content and scalability of the Optopatch synaptic assay, in addition to its applicability to human cultures, make it a powerful tool in resolving synaptic phenotypic responses in neurological diseases and screening candidate therapeutics.

8. In Vivo Multiphoton Imaging of Polymer Dot-Labeled Vasculature in C57 Mice. AHMED M. HASSAN,¹ JEREMY W. JARRETT,¹ DAVID R. MILLER,¹ YEN-LIANG LIU,¹ HSIN-CHIH YEH,¹ and ANDREW K. DUNN,^{1 1}Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX

In vivo bioimaging has emerged as the experimental foundation of many labs spanning a diverse range of research disciplines. Of the various approaches, multiphoton microscopy (MPM) is particularly well suited for deep, in vivo imaging due to its non-invasiveness and ability to maintain high spatial resolution at depth through a nonlinear dependence on excitation intensity. MPM demands bright, photostable contrast agents to overcome the strong absorption and scattering events that occur in biological tissue. Polymer dots (pdots) emerge as a highly emissive class of probes with promising characteristics and immense potential for the next generation of bioimaging. Here, we measure the two- and three-photon power dependence of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep neuroimaging of pdot-labeled mice. We find that all three pdots demonstrate a 2P power dependence across the titanium:sapphire tuning range and a 3P dependence at longer wavelengths using an optical parametric amplifier (OPA). In addition, we show that pdots’ action cross sections are one to two orders of magnitude brighter than quantum dots and three to five orders of magnitude larger than conventional fluorescent dyes and proteins in the near-infrared regime. Their extraordinary brightness enables 2P imaging of vasculature in mice at a depth of 850 µm with excellent SBR. We also find that pdots’ broad absorption confers compatibility with a high-power ytterbium-fiber (yb-fiber) laser and a high pulse energy OPA. Taking advantage of the unique properties of these laser systems, the intrinsic reduction of scattering of longer wavelengths, and the suppression of out-of-focus fluorescence introduced by higher order non-linear excitation, we extend imaging depth (z_yb-fiber = 900 µm, z_OPA = 1300 µm) and improve SBR substantially. With these brighter probes, researchers will be able to perform in vivo imaging with improved clarity and depth, enabling critical insights into fundamental biological problems.

9. High-Throughput FRET Assays for cAMP Using the Epac1-Based Biosensor H188 Enable GPCR Agonist Drug Discovery. GEORGE G. HOLZ, COLIN A. LEECH, and OLEG G. CHEPURNY, State University of New York Upstate Medical University, Syracuse, NY

High-throughput screening of small molecule libraries is one means by which to identify new pharmacological agents that activate G protein–coupled receptors (GPCRs) to stimulate cAMP production so that ligands with beneficial medicinal properties are discoverable. Here, we report a new microplate reader assay that allows high-throughput detection of cAMP with fast temporal resolution and wide dynamic range for cells treated with GPCR agonists. The assay uses clonal HEK293-H188 cells that express the genetically encoded biosensor H188 in which the cAMP-binding protein Epac1 is flanked by mTurquoise2Δ and ^cp173Venus-Venus FRET donor/acceptor fluorophores. Analysis of HEK293-H188 cell monolayers demonstrates that this assay is suitable to monitor the increase or decrease of cAMP levels that results when GLP-1, GIP, Glucagon, Adenosine-2B, TGR5, and NPY2R receptors are stimulated by their respective agonists. Accurate dose–response analysis is made possible by the nearly 10-fold greater ΔFRET that H188 exhibits when compared with the first-generation biosensor Epac1-camps. By adapting this assay to studies of cyclic nucleotide analog action in living cells, we also define the properties of Epac1 activators and inhibitors with great precision. Thus, this microplate reader FRET assay enables GPCR drug discovery, while providing a new platform with which to advance cyclic nucleotide research.

10. Red-to-Green Photoconversion of mCherry as a Source of Potential Artifacts in Multicolor Labeling Experiments. JASON Y. JIANG, JEFFREY L. FALCONE, SILVANA CURCI, and ALDEBARAN M. HOFER, Dept. of Surgery, VA Boston Healthcare System, Brigham and Women’s Hospital and Harvard Medical School

The monomeric red fluorescent protein mCherry from the soft coral Discosoma is considered among the most stable and rapidly maturing of the red fluorescent proteins (RFPs), accounting for its widespread popularity as a protein tag and constituent of genetically encoded sensors. We observed significant “greening” of mCherry and several other related RFPs upon illumination of the red fluorescence using power settings that are within normal limits for many types of fluorescence microscopy experiments (confocal and epifluorescence). Red-to-green behavior was observed when mCherry was expressed either alone or when fused to protein partners in several types of living cells, in fixed cells, and in droplets of purified protein over a wide range of protein concentrations. Although the green species that emerged after photobleaching was only about 10% as bright as mCherry, and dim compared to EGFP, we provide examples of how this characteristic can lead to significant artifacts during co-localization experiments employing green fluorescent proteins or when combined with genetically encoded calcium and cAMP sensors. Photoconverted mCherry thus has the potential to be confused with green FPs under many types of experimental conditions, for example, when GFP is used to report endogenous protein levels (as in transgenic animals or CRISPR knock-ins), or when combined with other green FPs that are not necessarily as bright as GFP. Our observations do not detract from the utility of mCherry, which remains one of the “best-behaved” RFPs available, but do highlight the importance of appropriate controls to assess the impact of red-to-green photoconversion in such situations.

11. Recording Neural Activity in Freely Behaving Animals With 3D Tracking Two-Photon Microscopy. DOYCHO KARAGYOZOV,¹ MIRNA MIHOVILOVIC SKANATA,¹ AMANDA LESAR,¹ and MARC GERSHOW,^1,2,3 New York University, Department of Physics¹, Center for Neural Science², Neuroscience Institute³

The Drosophila larva, a genetic model organism with a transparent cuticle and compact brain, is a nearly ideal substrate for optical neurophysiology. Optical recording of neural activity from behaving larvae has proved elusive due to rapid three-dimensional brain movements generated by peristaltic crawling. Recent developments in light sheet and extended depth of field microscopy show promise in imaging moving subjects, but these techniques can be limited by scattering in tissue, and the excitation light presents an unwanted visual stimulus as well as an obstacle to simultaneous optogenetic manipulation. Here, we present a two-photon microscope capable of high bandwidth fluorescent recordings in three-dimensionally moving neurons without motion artifacts. Using conventional galvonometric mirrors and a resonant ultrasonic lens, we move the microscope’s focal spot rapidly in a cylinder about the center of a single targeted neuron. We count the photons emitted from each point on the scan, use these to form an updated estimate of the neuron’s location, and execute the next scan around the updated center. The algorithm is implemented in FPGA hardware, and the feedback loop closes in 360 microseconds. A secondary feedback moves the microscope stage to keep the neuron centered in the field of view. Because the focal spot never leaves the neuron, we achieve a continuous recording of fluorescence with a bandwidth exceeding 1 kHz.

To demonstrate the utility of the microscope, we recorded from individual aCC motor neurons in the ventral nerve cord and correlated their activities with the larva’s peristaltic motion, visualized simultaneously on an infrared video camera. We then tracked visual interneurons and recorded their responses to blue light stimuli. Finally, we simultaneously tracked and recorded from two adjacent neurons. To our knowledge, this is the first two-photon recording of neural activity in which the brain was not rigidly coupled to the microscope.

12. Optogenetic Cre/loxP recombination system with high efficiency. FUUN KAWANO¹ and MASAYUKI YAZAWA,^{1,2 1}Department of Rehabilitation and Regenerative Medicine, Columbia University, New York, NY 10032; ²Department of Pharmacology, Columbia University, New York, NY 10032

Site-specific DNA recombination systems represented by Cre/loxP are a powerful tool for modifying transgenes in chromosomes of living organisms. A variety of conditionally inducible Cre/loxP recombination systems have been developed in combination with cell-, tissue-, or developmental stage–specific promoters and/or chemically inducible systems. However, such conventional approaches have relatively poor spatiotemporal resolution. Recently emerged optogenetic approaches are superior in terms of high tunability, rapid tissue penetration, and non-invasiveness in addition to high spatial and temporal confinement. However, powerful and useful Cre/loxP recombination techniques based on optogenetic systems remain elusive. Here, we describe a genetically encoded photoactivatable Cre recombinase (named PA-Cre) with high efficiency to implement DNA recombination with light in living systems (Kawano et al. 2016. Nat. Chem. Biol. 12:1059–1064). PA-Cre is based on reassembling of split Cre fragments driven by heterodimerization of nMag and pMag that are photoswitches recently engineered from a blue light-absorbing fungal photoreceptor Vivid (Kawano et al. 2015. Nat. Commun. 6:6256). We fused nMag and pMag with CreN59 (residues 19–59) and CreC60 (residues 60–343) of split Cre fragments, respectively. We optimized them in terms of linker amino acid sequences and configurations on the basis of structural information of Cre and the Vivid dimer in the light state. As a result of the step-by-step improvements, we obtained PA-Cre that yields a substantially efficient DNA recombination (up to 320-fold induction) with high spatiotemporal precision upon blue light illumination in vitro and in vivo. Additionally, by taking advantage of nMag and pMag having slow photocycle kinetics (t_1/2 = 1.8 h), we demonstrated that PA-Cre enables DNA recombination in vitro and in vivo even through illumination for only a short period of time (∼30 s). This robust technique will facilitate optogenetic genome engineering in living systems.

13. Adaptive Optics Confocal Microscopy for Voltage Imaging in Tissue and In Vivo. SIMON KHEIFETS, YOAV ADAM, DAAN BRINKS, and ADAM COHEN

Functional imaging using genetically encoded voltage indicators promises to enable new insights into neural dynamics. Potential applications include high-throughput multi-cell intracellular recording, as well as measurement of the propagation and compartmentalization of voltage within a neuron. Such measurements are hindered by the relatively low signal-to-noise ratio (SNR) currently achieved when imaging at millisecond time scales. Voltage imaging in intact mammalian brain tissue faces a unique combination of factors that negatively affect SNR. Besides the relatively low brightness and/or sensitivity of existing reporters, other confounding factors include the sparse, intertwined geometry of cell membranes, the high temporal bandwidth requirements, and the scattering, aberration and autofluorescence of mammalian brain tissue. The techniques that have been successful for calcium imaging are not optimal for voltage imaging. We have been developing microscopy techniques that optimize the SNR for voltage imaging in tissue. These techniques include aberration-corrected confocal microscopy for spatially resolved single-cell recordings, as well as holographic illumination for wide-field multi-cell recording. Preliminary results demonstrate robust optical recording of action potentials and subthreshold events in acute slice and in vivo.

14. Calcium Dynamics Regulating the Timing of Decision Making in C. elegans. KOTARO KIMURA, Osaka University

Brains process sensory information to generate various kinds of physiological responses with different timings (i.e., with different latencies to respond; Buhusi and Meck. 2005. Nat. Rev. Neurosci. 6:755–765). In decision making, for example, animals choose one from multiple behavioral options based on environmental sensory information, where a temporal delay is associated with the certainty of sensory information (Gold and Shadlen. 2007. Annu. Rev. Neurosci. 20:535–574). The neural mechanism of timing, however, is largely unclear. We report here the cellular and molecular mechanisms underlying the timing of decision making during olfactory navigation in Caenorhabditis elegans. We found that, based on subtle changes in concentrations of the repulsive odor, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. By analyzing neural responses in freely behaving animals under virtual odor gradients with calcium imaging, optogenetics, mathematical modeling, and genetics, we found that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca²⁺]_i), which occurs via L-type voltage-gated calcium channels (VGCC) in a pair of AWB olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca²⁺]_i rapidly increases via multiple types of calcium channels in a pair of ASH nociceptive neurons. Our data suggest that the timing of neuronal responses can be determined by cell type–dependent involvement of calcium channels and that a single-cell temporal integrator with L-type VGCCs, such as the AWB neuron, may be the evolutionarily conserved neural basis for decision-making.

15. Characteristics of the b2 Subunit of the Calcium-Activated K⁺ Channel in Skate Electroreceptors. BENJAMIN L. KING,¹ PETER KAO,² LING FANG SHI,² and WILLIAM T. CLUSIN,² University of Maine¹ and Stanford University Medical School²

Our comparative studies seek to understand the structure and function of ion channels through the study of cartilaginous fish electroreceptors that can detect voltage gradients as low as 5 nanovolts per centimeter. Existence of calcium activated K⁺ channels in excitable electroreceptor cells of the skate, L. erinacea, was first proposed in 1974. In humans, the large conductance (B_K) channel is comprised of four α-subunits, encoded by KCNMA1, and modulatory β-subunits, encoded by KCNMB1, KCNMB2, KCNMB3, and KCNMB4. We recently cloned the skate KCΝΜA1 gene using purified mRNA from homogenized isolated electroreceptors (King et al. 2016. Gene. 578:63–73). The skate KCNMB4 subunit was also partially cloned in that study. Recently, Bellono et al. (2017. Nature. 543:391–396) performed RNA sequencing (RNA-Seq) on purified mRNA from skate electroreceptors and found several ion channels including KCNMA1. Because the β-subunits play an important role in modulating B_K channel activity, we searched the RNA-Seq data for β-subunits and identified skate KCNMB2. The transcript encodes a 279-aa protein that contained 51 additional amino acids at the N terminus compared with human KCNMB2. The remaining 227 aa of the skate protein were 48% identical and 67% similar to human KCNMB2. Like the skate, alignment of the whale shark (R. typus) KCNMB2 showed 40 additional amino acids at the N terminus. Local alignment of the skate and whale shark proteins showed 56% identity and 72% similarity over 270 aa. KCNMB2 from a chimera, the elephant shark (C. milii), was 54% identical and 69% similar over 227 aa, an alignment length similar to human. As the N terminus ball-and-chain domain of human KCNMB2 is responsible for fast inactivation of B_K channels, the longer skate and whale shark N terminus may be functional. Further research should include confirmation by cloning and studies of how the gating behavior of skate B_K channels is modified by β-subunits.

16. Optogenetic Interrogation of Neural Inhibition with Light-Regulated GABA_A Receptors. WAN-CHEN LIN, MING-CHI TSAI, CHRISTOPHER M. DAVENPORT, and RICHARD H. KRAMER, Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720

Optogenetics is a hybrid approach that integrates optical and genetic manipulations of a biological target for precise and specific control in a complex system. Here we present the development and applications of optogenetic tools for unlocking the functional diversity of inhibitory neurotransmission in the brain. GABA_A receptors (GABA_ARs), a family of ligand-gated chloride channels, are major mediators of neural inhibition. A GABA_AR typically comprises two α, two β, and one tertiary subunit (γ or δ), with the α-subunit primarily determining its function and distribution. The expression profile of GABA_ARs is highly diverse across different cell types, brain regions, and developmental stages. The functional rationales underlying this tremendous diversity remain elusive. To unravel the roles of individual subtypes in various brain functions, we have employed a chemical-genetic approach to engineer light-regulated GABA_A receptors (LiGABARs) for all six α-isoforms, providing a comprehensive toolkit for optogenetic interrogation of neural inhibition. These receptors are bi-stable devices: they can be rapidly and reversibly switched between normal and antagonized states with two different wavelengths of illumination, and their functional states are preserved in darkness following a brief flash of conditioning light. The engineered receptors can be introduced into neurons exogenously via viral transduction or endogenously in knock-in mice. LiGABARs have been successfully implemented in brain slices and in vivo, enabling optical manipulation of inhibitory synaptic responses, neuronal firing, and network activities in the mouse brain. We have employed this toolkit to functionally probe the differential contribution of a1-GABA_ARs to distinct classes of inhibitory synapses in the mouse visual cortex. Moreover, we have also revealed the unique actions of α5-GABA_ARs in regulating hippocampal long-term plasticity. These results demonstrate the unprecedented potential of LiGABAR-based optogenetics in decoding inhibitory neurotransmission underlying brain physiology and disorders.

17. Decreased Ca²⁺ Signaling, Ca_V1 Channel Expression, and Activity in Pancreatic β Cells Expressing Truncated DISC1 (Disrupted in Schizophrenia 1). PING LU,¹ RICKY DAVID JONES,¹ RONGHUA ZHUGE,¹ ANN R. RITTENHOUSE,¹* and AGATA JURCZYK,²* ¹Department of Microbiology and Physiological Systems and ²Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA (*Senior Co-authors)

Schizophrenics often suffer from non-obese type 2 diabetes (T2D) in addition to cognitive impairment. In this study, we interrogated whether similar cellular changes occur in pancreatic β cells as found in neurons from a mouse model of schizophrenia. Specifically, we tested whether β cells exhibit disrupted excitation–secretion coupling when a truncated human DISC1 (thDISC1) gene, originally discovered in a Scottish family having high penetrance for schizophrenia (St Clair et al. 1990. Lancet. 336:13–16), is expressed selectively in mouse β cells upon ingesting doxycycline (+DOX). Previously, we found that expression of thDISC1 significantly decreases blood insulin levels and glucose-stimulated insulin secretion (GSIS) from isolated islets, revealing an independent role for DISC1 in β cells (Jurczyk et al. 2015. FASEB. J. 30:983–993). In central neurons, DISC1 regulates Ca_V2 channel expression, Ca²⁺ influx, and transmitter release (Tang et al. 2016. Front. Synap. Neurosci. http://dx.doi.org/10.3389/fnsyn.2016.00015). To determine whether DISC1 regulates Ca²⁺ physiology in β cells, we tested thDISC1 DOX+ versus DOX- β cells for changes in (i) intracellular Ca²⁺signaling using Fluo 3-AM; (ii) Ca_V1 channel expression, the dominant channel class controlling GSIS, using immunohistochemistry; (iii) and Ca_V1 channel activity using whole-cell recording methods. We found increases in [Ca²⁺]_i following 20 mM glucose were delayed >2 min and diminished ∼50% in dissociated β cells expressing thDISC1. When sections of pancreas were stained with an anti-Ca_V1.2/1.3 antibody (NeuroMab), thDISC1 DOX+ versus DOX- insulin-positive cells exhibited significant loss of staining. Consistent with lower functional Ca_V1 expression, both peak inward Ba²⁺ current, and FPL 64176–induced long-lasting L-type tail current decreased significantly in thDISC1 DOX+ (56 and 67%, respectively) versus DOX- β cells. No obvious change in activation kinetics was observed. These changes in excitation-secretion coupling mirror those in neurons expressing truncated DISC1. Thus, at the cellular level, truncated DISC1 precipitates disorders of secretion.

18. Visualizing the Arterial Wall Water Permeability Barrier with Femtosecond CARS Microscopy. BERTRAND M. LUCOTTE,¹ CHLOE POWELL,² JAY R. KNUTSON,³ KIM A. DORA,² and ROBERT S. BALABAN,^{1 1}Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD; ²Vascular Pharmacology Research Group, Department of Pharmacology, University of Oxford, Oxford, UK; ³Laboratory of Optical Spectroscopy, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD

A large arterial transmural pressure gradient is essential to the normal physiological function of the circulation and indicates the presence of a significant barrier to water permeation. The nature of this barrier has generated high research interests since increases in arterial wall water permeability are associated with many diseases, including diabetes, atherosclerosis, acute shock and inflammatory conditions, and the aging process. Despite the importance of this fundamental arterial wall property in vascular function and drug pharmacodynamics, the precise location and biochemical composition of the arterial wall water barrier is unknown. Many have suggested this barrier is formed by the endothelial cell (EC) layer based on the effect of its gross mechanical disruption.

Using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion, we directly map the water permeability, and the associated pressure profile, in in vitro intact and pressurized rat mesenteric arteries (Lucotte et al. 2017. Proc. Natl. Acad. Sci. USA. 114(18): 4805–4810). Unlike most metabolites found in biological tissues, water CARS imaging at the OH stretch resonance is best achieved with femtosecond rather than picosecond excitation pulses. The signal thus generated is increased by two orders of magnitude while retaining isotopic specificity.

We identify the endothelial basolateral membrane as the major arterial water barrier and show that the apical membrane is highly permeable. This is confirmed by the endothelial distribution of the AQP1 water channel, which is found exclusively at the apical membrane. The advantageously positioned water barrier permits (1) arterial pressure to equilibrate within the EC cytosol and (2) direct arterial pressure transfer to the supporting basement membrane and internal elastic lamina macromolecules with minimal EC deformation. This transfer of pressure thus provides the mechanically sensitive EC with a protective mechanism against the recurring arterial pressure waves. Its disruption could contribute to EC dysfunction in various pathologies.

19. Effects of Peptidoglycan on Localization and Function of TonB in Escherichia coli. ARITRI MAJUMDAR, SALETE M. NEWTON, and PHILLIP E. KLEBBA, Department of Biochemistry and Molecular Biophysics, Kansas State University

Iron uptake through Gram (-) bacterial outer membrane (OM) proteins occurs by active transport that requires energy-transducing activity of the inner membrane (IM) protein, TonB. OM transporters like FepA bind ferric complexes and import them as a result of protein–protein interactions with TonB, which undergoes energy-dependent motion in the IM. TonB also binds the peptidoglycan (PG) beneath the OM, which organizes biogenesis/physiology and defines cell shape. This fact may explain restricted distribution of TonB to the central region of Gram (-) cells. We microscopically observed fluorescently labeled FepA and TonB proteins in vivo and correlated these images with radioisotopic iron uptake experiments in normal E. coli and in cells with compromised, degraded PG. The results showed that as PG was enzymatically degraded from within, GFP-TonB lost its restricted, central localization, and uniformly distributed throughout the IM. Degradation of PG reduced or abrogated uptake of [⁵⁹Fe]enterobactin through the OM, without compromising the uptake of [¹⁴C]lactose through the IM, despite the fact that both transport reactions depend on electrochemical proton-motive force (PMF). These data revealed that restricted distribution of TonB in the central part of the cell, and its exclusion from the poles of the cell, derives from its association with the PG polymer in the bacterial periplasm. Furthermore, degradation of PG undermines TonB-dependent transport of ⁵⁹Fe through the OM, suggesting that associations between TonB and PG are mechanistically crucial to the function of OM metal transporters like FepA. We interpret the results in the context of the ROSET model of TonB-dependent active iron OM transport, which postulates low-affinity binding of the TonB C terminus to both PG and OM proteins, which, in combination with PMF-driven rotational motion, facilitates mechanical energy transfer from IM to OM. These reactions allow active transport in a bilayer that cannot sustain an ion gradient.

20. The Thresholds of an Excitable Signal Transduction Network Determine Cell Migratory Modes. YUCHUAN MIAO,¹ SAYAK BHATTACHARYA,² MARC EDWARDS,³ HUAQING CAI,⁴ TAKANARI INOUE,³ PABLO A. IGLESIAS,^2,3 and PETER N. DEVREOTES,^{3 1}Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; ²Department of Electrical and Computer Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD 21205; ³Department of Cell Biology and Center for Cell Dynamics, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; ⁴State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

The diverse migratory modes displayed by different cell types are generally believed to be idiosyncratic. Here, we show that the migratory behavior of Dictyostelium was switched from amoeboid to keratocyte-like and oscillatory modes by synthetically decreasing PIP2 levels or increasing Ras/Rap-related activities. The perturbations at these key nodes of an excitable signal transduction network initiated a causal chain of events: The threshold for network activation was lowered, the speed and range of propagating waves of signal transduction activity increased, actin-driven cellular protrusions expanded, and, consequently, the cell migratory mode transitions ensued. Conversely, innately keratocyte-like and oscillatory cells were promptly converted to amoeboid by inhibition of Ras effectors with restoration of directed migration. We propose a unifying theory of how and why cells move, which might explain migratory transitions found in development as well as in pathological conditions.

21. In Vivo AO-SLO/OCT Imaging of Microglia During the Initiation and Resolution of Retinal Degeneration. ERIC B. MILLER, PENGFEI ZHANG, EDWARD N. PUGH, JR. and MARIE E. BURNS

Microglia, the resident macrophages of the central nervous system, are thought to be highly dynamic ramified cells that constantly surveil their microenvironment to remove neuronal debris and prune synaptic contacts. Using custom, high-resolution dual scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) systems with and without adaptive optics (AO), we have developed a highly repeatable in vivo preparation that allows high-resolution visualization of microglial activation, migration, and de-activation in response to localized, transient neuronal damage. In healthy retina, AO-SLO time-lapse imaging of GFP-expressing microglia (Cx3CR1^+/GFP mice) revealed strikingly little movement of the primary microglial branches, even 1 hour after an infrared light exposure that produced a spot (∼150 mm) of acute damage to rod photoreceptors, as determined from changes in OCT light-scattering in the outer nuclear layer. However, over the course of days following the damaging exposure, microglia lost their branched structure and migrated in 3D to the region of damage, densely populating the area while other microglia only a few hundred micrometers away remained highly ramified and stationary. Gradually, microglia at the damage locus returned to their normal ramified morphology and mosaic distribution after 2 weeks. The return of microglia to their normal state coincided with the disappearance of light scattering changes associated with damaged rods. These results demonstrate ramified microglia in both healthy and damaged retina are relatively stationary rather than highly dynamic and that dramatic functional variation in subsets of microglial cells depend on their specific location within the CNS. Furthermore, the ability to image both microglial activation, migration and resolution in vivo opens the door to visualizing the signaling mechanisms underlying these dramatic cellular transformations that are thought to be central for synaptic plasticity and maintenance, as well as mitigation of neurodegenerative diseases.

22. Stoichiometry of Kv2.1/Kv6.4 Heterotetramers Revealed by Fluorescent Single Channel Subunit Counting. LENA MOELLER,¹ GLENN REGNIER,² ALAIN J. LABRO,² DIRK J. SNYDERS,² and RIKARD BLUNCK,^{3 1}Department of Biochemistry, Université de Montréal, Montreal, Canada; ²Biomedical Sciences, University of Antwerp, Antwerpen, Belgium; ³Department of Physics, Université de Montréal, Montreal, Canada

It was previously shown that electrically silent voltage-gated channel subunits (KvS) do not form functional homotetramers but can assemble as heteromeric Kv2/KvS channels with rendered biophysical properties compared with Kv2 homotetramers. So far, only few of these heteromeric channels have been studied regarding their stoichiometry. FRET measurements have shown that for example Kv2.1/Kv9.3 channels have a fixed stoichiometry of 3:1 (Kerschensteiner et al. 2005. Proc. Natl. Acad. Sci. 102(17):6160–6165). In order to assess whether other heteromeric Kv2.1/KvS channels also adopt this arrangement of subunits, we previously investigated the Kv2.1/Kv6.4 heteromer by examining the functional channel properties of different concatemers. We found that the stoichiometry in these heteroteramers can be either 3:1 or 2:2. Here, we co-express Kv2.1 and a fluorescently tagged Kv6.4-GFP in Xenopus oocytes and verify the stoichiometry by single-molecule fluorescence imaging. Co-expressed Kv6.4-GFP is subjected to a fluorescence imaging. The number of fluorescently labeled subunits within a single heteromer can then be determined by counting the photobleaching steps observed as progressively each fluorescent label loses its fluorescence in a stepwise distinct event by photochemical destruction (Ulbrich and Isacoff. 2007. Nature Methods. 4(4):319–321). We confirm that there are maximally two Kv6.4 subunits per heteromeric complex. Furthermore, our results suggest that this arrangement of subunits is independent of the expression level of Kv6.4 subunits in the cell.

23. Finding Two-Photon Brighter Green Fluorescent Proteins in Obscure, Blue-Shifted Variants. ROSANA S. MOLINA,¹ MERRILEE THOMAS,¹ TAM M. TRAN,² GERARD G. LAMBERT,³ ANYA SALIH,⁴ ROBERT E. CAMPBELL,² NATHAN C. SHANER,³ THOMAS HUGHES,¹ and MIKHAIL DROBIZHEV,^{1 1}Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana 59717; ²Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada; ³Scintillon Institute, San Diego, California 92121; ⁴Western Sydney University, Penrith South DC, New South Wales 1797, Australia

Two-photon microscopy together with genetically encoded fluorescent biosensors has facilitated deeper imaging of living tissue, especially the brain, leading to insights into physiological processes based on markers such as calcium activity and voltage changes across the membrane. Fluorescent proteins (FPs) and their physical properties are a critical consideration when designing new biosensors. Enhanced two-photon brightness (defined as the two-photon cross section multiplied by the fluorescence quantum yield) is particularly desirable because the high intensities of the two-photon excitation laser can cause tissue damage: with a brighter sensor, a lower laser power can be used for an equivalent signal. Genetic mutations within the beta barrel of an FP can cause the electrostatics around the chromophore to change, impacting both the one-photon and two-photon properties. Expanding on previous physical modeling of the anionic green FP (GFP) chromophore (Drobizhev et al. 2012. J. Phys. Chem. B. 116:1736–1744; Drobizhev et al. 2015. Sci. Rep. 5:13223), we derived a relationship that predicts a stronger two-photon maximum cross section with a blue-shifted one-photon absorption. In accordance with this prediction, we measured 12 blue-shifted FPs with this chromophore and found that they are up to two and half times brighter than the commonly used enhanced GFP, whereas the red-shifted but one-photon bright mNeonGreen is two times dimmer (Molina et al. 2017. J. Phys. Chem. Lett. 8:2548–2554). Our model and experimental data suggest that FPs should be optimized independently for the two-photon regime. Progress is underway to screen for even brighter blue-shifted mutants.

24. Opsin 3–Mediated Regulation of Skin Pigmentation. LAUREN E. OLINSKI,¹ RANA N. ÖZDEŞLIK,² and ELENA OANCEA,^{2 1}Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI; ²Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI

Opsins—light-activated G protein–coupled receptors (GPCRs)—are crucial junctions bridging photostimulation with internal signaling events. As photoreceptors, the majority of vertebrate opsins have been extensively characterized (Terakita. 2005. Genome Biol. 6:213), yet the functionality of extraocular opsin 3 (OPN3) remains to be elucidated. OPN3, first discovered in the mammalian brain (Blackshaw and Snyder. 1999. J. Neurosci. 19:3681–3690), is different from other vertebrate opsins in that it is expressed in several peripheral tissues, many of which are not readily exposed to light (Halford et al. 2001. Genomics. 72:203–208), and possesses a long carboxy terminus with no homology to other GPCRs. We recently discovered that OPN3 is highly expressed in human epidermal melanocytes (HEMs; Haltaufderhyde et al. 2015. Photochem. Photobiol. 91:117–123), the melanin-producing cells of the skin. We found that reducing OPN3 expression levels in HEMs leads to an increase in melanin production, linking OPN3 to melanogenesis. Intriguingly, in HEMs, OPN3 interacts with melanocortin-1 receptor (MC1R), a key regulator of the melanogenic pathway. Activation of MC1R, a G_as-coupled GPCR (Yamaguchi et al. 2007. J. Biol. Chem. 282:27557–27561), leads to increased cAMP levels, while OPN3, a predicted G_ai-coupled GPCR (Koyanagi et al. 2013. Proc. Natl. Acad. Sci. USA. 110:4998–5003), has the opposite effect on cAMP levels, suggesting that the mechanistic purpose underlying the interaction of OPN3 and MC1R may hinge on regulation of cAMP levels upstream of melanin production. While invertebrate and non-mammalian homologues of OPN3 have been shown to be activated by blue light (Koyanagi et al. 2013. Proc. Natl. Acad. Sci. USA. 110:4998–5003), we did not detect light-induced OPN3 signaling in HEMs, suggesting that in these cells OPN3 may function as a constitutively active receptor or is activated by very low levels of ambient light. Our data reveal a novel function for the poorly characterized human OPN3 in skin pigmentation.

25. Two-Photon Imaging of Neuronal Activity in Freely Behaving Mice Using a Miniature Fiber-Coupled Microscope with Active Axial Focusing. BARIS N. OZBAY, GREGORY L. FUTIA, MING MA, ETHAN G. HUGHES, DIEGO RESTREPO,* and EMILY A. GIBSON,* University Of Colorado Denver, Aurora, CO; *Senior Co-authors

We have designed and built a miniature two-photon fiber-coupled microscope (TP-FCM) with active axial focusing for imaging neuronal activity in behaving and freely-moving mice. The TP-FCM has a weight of ∼4 g. A coherent fiber-bundle with a diameter of 0.5 mm is used as a lateral scanning relay for excitation and emission collection, allowing the use of bench-top scanning and detection optics with multiple fluorescent indicators. A miniature electrowetting tunable lens enables active axial focusing. Two versions of the TP-FCM are demonstrated. (1) A cortical imaging TP-FCM with a cylindrical imaging volume of 200-µm diameter × 180-µm depth, lateral resolution of ∼1.8 µm, and axial resolution of ∼10 µm, designed for imaging through a glass cranial window. (2) A deep-brain TP-FCM designed, with an imaging volume of ∼150-µm diameter × 100-µm depth, lateral resolution of ∼1.4 µm, and axial resolution of ∼8 µm, designed for imaging through a 0.5-mm diameter GRIN lens. We show neuronal activity recorded from the motor cortex neurons expressing GCaMP6s of freely behaving mice. The TP-FCM design allows for the recording of neuronal activity from a large three-dimensional volume with the ability to rapidly change axial focus and execute more arbitrary volumetric scan trajectories.

26. Distance-Resolving Voltage Clamp Fluorometry: Ångström-Precision Measurements of Membrane Protein Structural Dynamics Under Physiological Conditions. ANTONIOS PANTAZIS and RICCARDO OLCESE, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine, David Geffen School of Medicine, University of California, Los Angeles

The holy grail of structural biology, i.e., the simultaneous quantitative determination of a protein’s structure and function, remains very difficult to attain. Voltage clamp fluorometry (VCF) is an optical approach for characterizing the functional properties of fluorescently labeled domains of ion channels and related proteins. We adapted both experimental and theoretical aspects of VCF, endowing it with the ability to accurately measure intramolecular distances up to 2.5 nm with a very fine grain. We call the new approach distance-resolving VCF (drVCF).

Using drVCF on conducting human BK channels expressed in oocytes under voltage clamp, we found that the transmembrane helix S4 of the voltage-sensing domain (VSD) is virtually equidistant from juxtaposed helices S1 and S2 in the Active state (mean, [95% confidence intervals]; S1–S4: 16.5 [15.5,17.5] Å; S2–S4: 15.9 [15.1,17.5] Å). These measurements are in excellent agreement with the open BK channel structure recently resolved by cryo-EM (Tao et al. 2017. Nature. 541:46–51; S1–S4 and S2–S4 distance ∼16 Å). We also found that, in the resting state, S4 is nearer to S1 and S2 (7.5 [5.7,9.3] and 7.1 [6.0,8.6] Å, respectively). This information on the rearrangement of S4 with respect to S1 and S2 provides the structural correlate for depolarization-evoked BK channel activation and, as it was acquired from functional, conducting channels, an ideal complement to BK functional studies.

To validate drVCF, we used synthetic polyproline-based peptides, initially employed by Stryer and Haugland to validate FRET. In peptides with 2, 4, or 7 prolines, drVCF length estimates were off, on average, by 0.99 ± 0.24 Å, also providing a good estimate for the per-residue rise of a polyproline type II helix (drVCF: 3.4 [3.0,3.7] Å; actual: 3.1 Å). Thus, drVCF is a remarkably accurate optical approach for the real-time measurement of intramolecular distances and distance changes under physiologically relevant conditions, simultaneously with protein function.

27. A Photoconvertible Genetically Encoded Glutamate Highlighter for Astrocyte Imaging. STELIOS PAPADOPOULOS,¹ GRACE OR,¹ JENNIFER JAHNCKE,² CHUNYANG DONG,¹ JASON LAMBERT,² KAREN ZITO,² and LIN TIAN,^{1 1}Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, Davis, CA; ²Center for Neuroscience, University of California, Davis, Davis, CA

In the last decade, astrocytes have emerged as key participants in brain development and function in health and disease (Volterra and Meldolesi. 2005. Nat. Rev. Neursci. 6(8):626–640). Astrocytes have shown to play a crucial role in synaptogenesis and synaptic plasticity (Perea et al. 2009. Cell. 32(8):421–431) by responding to neurotransmitters (Porter and McCarthy. 1996. J. Neurosci. 16(16):5073–5081) and by releasing gliotransmitters (Araque et al. 2014. Cell. 81(4):728–739). However, the mechanisms through which astrocytes interact with neurons and contribute to the structural and functional plasticity of the brain remain poorly understood (Haber et al. 2006. J. Neurosci. 26(35):8881–8891; Clarke and Barres. 2013. Nat. Rev. Neurosci. 14(5):311–321). Recently, improved genetically encoded indicators of neuronal activity have allowed for functional measurements through optical recordings of calcium (Tian et al. 2009. Nat. Methods. 6(12):875–881) and glutamate (Marvin et al. 2013. Nat. Methods. 10(2):162–170). These applications have significantly advanced the field of systems neuroscience by permitting optical recordings in neurons, but have been inadequate in the context of astrocytes. The reason for this is that until recently we lacked an optical approach that can provide both a super-resolution image of the subcellular distribution of astrocytic compartments and a functional readout. Therefore, we have developed a new class of genetically encoded photo-convertible glutamate highlighters that permit functional recordings of glutamate transients and post-hoc analysis of the distribution of glutamate transporters, correlating functional plasticity with structural plasticity. We demonstrate the utility of this new glutamate highlighter in primary astrocyte neuron co-cultures. Ultimately, we expect this tool to bridge the knowledge gap of the constant interplay between neurons and astrocytes contributing to the plasticity of the brain.

28. Imaging Neuromodulatory Signaling With Genetically Encoded Indicators. TOMMASO PATRIARCHI,¹ KOHEI J. SEKIGUCHI,² AARON MARLEY,³ BRIAN P. MCGREW,¹ JOHN T. WILLIAMS,⁴ MARK VON ZASTROW,³ AXEL NIMMERJAHN,² and LIN TIAN,^{1 1}Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA; ²Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, CA; ³Department of Psychiatry, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA; ⁴Vollum Institute, Oregon Health and Sciences University, Portland, OR

Neuromodulation is a key feature of all nervous systems (Marder. 2012. Neuron. 76:1–11). Neuromodulators such as dopamine, norepinephrine, or serotonin exert powerful control over neural circuit dynamics linked to, for example, arousal, attention, emotion, or cognitive perception. Altered neuromodulator signaling is a key feature of virtually all human neurological and psychiatric disorders, including Parkinson’s disease, schizophrenia, depression, and addiction (Tye and Deisseroth. 2012. Nat. Rev. Neurosci. 13:251–266). We address the need for large-scale in vivo optical measurement of neuromodulator signaling by developing a toolbox of genetically encoded indicators based on circularly permuted GFP for a variety of neuromodulators including dopamine, norepinephrine, and serotonin. We have focused on characterizing the sensitivity, affinity, and specificity of these sensors in detecting dopamine and norepinephrine in mammalian cells and in dissociated neurons. In addition, we further tested the performance of these sensors in acute brain slices and in behaving mice with regard to their ability to detect stimulated or behaviorally evoked monoamine release. We expect these new tools to be broadly applicable, permitting sensitive measurement of neuromodulator signaling at synaptic, cellular, and system levels, furthering our understanding of fundamental neural circuit function.

29. Source-Localized Multifocal Two-Photon Microscopy for High-Speed Functional Imaging. PETER QUICKE,^1,4 MARK NEIL,^2,4 THOMAS KNÖPFEL,^3,4 SIMON R. SCHULTZ,^1,4 and AMANDA J. FOUST,^{1,4 1}Department of Bioengineering, ²Department of Physics, ³Department of Medicine, ⁴Centre for Neurotechnology, Imperial College London

We have optimized a multifocal two-photon microscope (MTPM) for high-speed functional imaging of neuronal activity. Accurately reconstructing underlying cellular dynamics from recorded fluorescence indicator traces requires high temporal and spatial resolution imaging. Two-photon point scanning (TPPS) approaches allow deep imaging in scattering tissue like the brain but are limited in temporal resolution by the need to serially acquire each image pixel. Parallelizing this acquisition by using multiple foci allows a large increase in speed whilst fully sampling the field of view. The need to image the resulting fluorescence, however, means that this technique is vulnerable to image blur by emission photon scattering. We have implemented a spatially sparse MTPM that can resolve cellular-resolution calcium transients in adult mouse brain slices bulk loaded with Cal-520. Imaging at 200 frames per second with a 110 × 200–µm field of view, the system resolves calcium transients with a signal to noise ratio of 10 ± 2 dB. We have also implemented image reconstruction algorithms that take advantage of the structured illumination in our sample to reduce the effect of scattering and we quantify the performance improvement in terms of signal localization, depth penetration, and contrast enhancement. We assess our system performance compared with TPPS and wide field single photon imaging. We also discuss the limiting factors of the system, in particular imaging depth and available laser power, which will determine the broader applicability of this technique.

30. Relationship Between Different Short-Term Plasticity Mechanisms at an Identified Synapse. MICHELLE A. SÁNCHEZ-RIVERA, MONTSERRAT G. CERCÓS, and CITLALI TRUETA, Departamento de Neurofisiología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Mexico City, Mexico

Short-term plasticity changes the amount of neurotransmitter release and thus the amplitude of postsynaptic potentials (PSP) at chemical synapses, depending on the stimulation frequency. An increase in the PSP amplitude, known as facilitation, is caused by an increase of the release probability due to presynaptic calcium accumulation. A decrease in the PSP amplitude after repetitive stimulation, known as depression, is attributed to presynaptic vesicular depletion. At some synapses depression occurs upon pairs of impulses at low frequencies, while paired impulses at higher frequencies produce facilitation, which is not congruent with the hypothesis of vesicular depletion. Here, we investigated the mechanisms that produce this type of depression at the synapse between pressure-sensitive and anterior pagoda neurons of the leech central nervous system. Presynaptic impulses separated by variable intervals were produced, and plasticity was analyzed by comparing the amplitudes of the resulting PSP. Two presynaptic impulses separated by 200 ms produced facilitation in the second PSP, which gradually decreased as the interval increased. Intervals larger than 800 ms produced depression. Increasing the initial transmitter release by rising extracellular calcium or by producing more impulses increased depression, suggesting a mechanism mediated by vesicular depletion. However, facilitation could be produced after a depressed PSP, indicating that there are enough synaptic vesicles at the terminal to sustain release and suggesting an additional depression mechanism, which depends on the interval between impulses. A plasticity mathematical model that considers vesicular depletion and changes in release probability due to intracellular calcium reproduces facilitation after short intervals, but not depression after long intervals, showing that known mechanisms do not explain this plasticity phenomenon. We propose that, in addition to vesicular depletion, there is a mechanism acting on vesicle availability whereby an impulse prepares vesicles to fuse with the membrane, but this is reversed after 800 ms, preventing their fusion.

31. Monitoring Voltage Fluctuations of Internal Cell Membranes. MASOUD SEPEHRI RAD,¹ LAWRENCE B. COHEN,^1,2 O. BRAUBACH,¹ and BRADLEY J. BAKER,^{1,3 1}Center for Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, 02792, Korea; ²Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520; ³Department of Neuroscience, Korea University of Science and Technology, Daejeon, South Korea

In eukaryotic cells, the endoplasmic reticulum (ER) is the largest continuous membrane-enclosed network that surrounds a single lumen. Using a new genetically encoded voltage indicator (GEVI), we applied the patch clamp technique to HEK293 cells and found that there is a very fast electrical interaction between the plasma membrane and internal membrane(s). This discovery suggests a novel mechanism for interaction between the external membrane and internal membranes as well as an additional mechanism for interactions between the various internal membranes. The ER may transfer electrical signals between the plasma membrane, the Golgi apparatus, the nuclear envelope, the mitochondria, and other internal organelles. The internal membrane signal is reversed in polarity but has a time course similar to that of the plasma membrane signal. The optical signal of the GEVI in the plasma membrane is consistent from trial to trial. However, the internal signal decreases in size with repeated trials. This dynamic behavior of the internal signal suggests that voltage may stress internal membranes causing them to remodel and/or change their resistance.

32. Nicotine in the Endoplasmic Reticulum. AMOL V. SHIVANGE,^1,2 AARON L. NICHOLS,² PHILIP M. BORDEN,¹ ARON KAMJAYA,² ANAND K. MUTHUSAMY,² JANICE H. JEON,² ELIZABETH K. UNGER,³ LIN TIAN,³ JONATHAN S. MARVIN,¹ LOREN L. LOOGER,¹ and HENRY A. LESTER,^{1,2 1}Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA; ²Division of Biology and Biological Engineering, Caltech, Pasadena, CA; ³Dept. of Biochemistry and Molecular Medicine, University of California at Davis

Nicotine activates plasma membrane (PM) nicotinic receptors (nAChRs), but also permeates into the endoplasmic reticulum (ER) and cis-Golgi, and there binds to nascent nAChRs. Other psychiatric and abused drugs may also enter the ER and bind their classical targets. Further progress requires direct proof, quantification, and time resolution of these processes in live cells and in the brain of animals. Therefore, we are developing genetically encoded fluorescent biosensors to study the subcellular pharmacokinetics of neural drugs.

OpuBC, a monomeric bacterial periplasmic binding protein (PBP), has (a) a binding site for amines including a cation-π box and (b) ligand-induced “Venus flytrap” conformational change involving relative motions of two domains—features reminiscent of Cys-loop receptors. We insert circularly permuted “superfolder” GFP (cpGFP), flanked by several-residue linkers, within inter-domain hinge regions. We apply directed evolution, including X-ray crystallography, to optimize the sensing of drugs, and have achieved the goal of ΔF/F0 > 1 at 1 µM for several drug-biosensor pairs.

Our most detailed studies use “intensity-based nicotine-sensing fluorescent reporters” (iNicSnFRs). We insert targeting and retention sequences to direct the constructs to the ER or to the PM of clonal mammalian lines and cultured neurons. Live-cell video imaging shows that, after we jump [nicotine] (increase or decrease) near cells, the drug appears in (or leaves) the ER within <10 s. Responses are robust, even at [nicotine] in the brain of a smoker. When extrapolated to human smoking and vaping, these data explain aspects of nicotine addiction. Kinetics with varenicline are nearly as fast, rationalizing its action in smoking cessation.

Other iDrugSnFRs detect antidepressants, opioids, and antipsychotics. We hope that further application of these tools will show the initial steps in the pathway of pharmacological chaperoning and its sequelae—aspects of “inside-out” neuropharmacology.

Support: NIH, NARSAD, California TRDRP, and HHMI.

33. Imaging Endogenous Neuronal Kv2 Potassium Channel Gating With a Fluorescent Tarantula Toxin. ROBERT G. STEWART,¹ PARASHAR THAPA,¹ REBECKA SEPELA,¹ OSCAR VIVAS,^1,2 LAXMI K. PARAJULI,² MARK W. LILLYA,¹ SEBASTIAN FLETCHER-TAYLOR,¹ BRUCE E. COHEN,³ KAREN ZITO,² and JON T. SACK,^{1 1}Physiology and Membrane Biology, University of California, Davis; ²Center for Neuroscience, University of California, Davis; ³Biological Nanostructures Facility, Laurence Berkeley National Laboratory

Neurons display diverse electrical signaling patterns throughout the nervous system. This electrical signaling is generated by voltage-gated ion channel subtypes working in concert. The localization and gating of channel subtypes determine how they contribute to neuronal signaling. However, it is challenging to establish where endogenous channel subtypes are expressed in live tissue and how active the channels are. We have generated molecular probes that optically report changes in the voltage activation status of Kv2 ion channels. These probes were developed from the tarantula peptide guangixitoxin (GxTX) that binds to resting Kv2 ion channels and disassociates when channels are activated by voltage. When GxTX is conjugated to fluorescent dyes, binding and disassociation can be visualized with fluorescence microscopy. By monitoring changes in fluorescence of a GxTX variant with 2-photon microscopy, we can observe voltage activation of endogenous Kv2 channels in CA1 neurons in rat hippocampal slices. These voltage-dependent changes from neurons are consistent with GxTX fluorescence responses from heterologously expressed Kv2.1 channels. The GxTX probe appears selective for Kv2 channels. In neurons and CHO cells, transfected with GFP-Kv2.1 or GFP-Kv2.2, GxTX puncta colocalize with GFP puncta. Colocalization with other GFP-tagged channel subtypes was not detected. Kv2 channel binding was retained in the presence of its neuronal auxiliary subunit AMIGO-1. We conclude that optical signals from conformation-selective ion channel probes can report when and where an endogenous ion channel subtype activates in individual neurons within a brain slice.

34. Defining Olfactory Bulb Functions via Comparison of Input and Output. DOUGLAS A. STORACE,¹ SHENG ZHONG,¹ and LAWRENCE B. COHEN,^{1,2 1}Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520; ²Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea

Humans and other animals can recognize an odorant as the same over a range of odorant concentrations. It remains unclear whether the olfactory bulb, the brain structure that mediates the first stage of olfactory information processing participates in generating this perceptual concentration invariance. Olfactory bulb glomeruli are regions of neuropil that contain input and output processes: olfactory receptor neuron nerve terminals (input) and mitral/tufted cell apical dendrites (output). Differences between the input and output of a brain region define the function(s) carried out by that region. We compared the activity signals from the input and output across a range of odorant concentrations. The output maps maintained a relatively stable representation of odor identity over the tested concentration range, even though the input maps and signals changed markedly. The results were similar when performing the measurements in the same preparation using different optical sensors, or in different preparations using the same sensor. These results provide direct evidence that the mammalian olfactory bulb likely participates in generating the perception of concentration invariance of odor quality. Our imaging methods should also be useful for determining the input/output transformation in other regions of the mammalian brain.

Supported by: US NIH Grants DC005259, DC016133, NS099691 and a Ruth L. Kirschstein National Research Service Award DC012981, Grant WCI 2009-003 from the National Research Foundation of Korea, and a James Hudson Brown–Alexander Brown Coxe Fellowship from Yale University.

35. A Second-Generation SCAPE System for Fast, 3D Imaging of Neural Activity. VENKATAKAUSHIK VOLETI, WENZE LI, KRIPA PATEL, MOHAMMED A. SHAIK, CITLALI PEREZ-CAMPOS, and ELIZABETH M.C. HILLMAN, Departments of Biomedical Engineering and Radiology, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University in the City of New York, NY 10027

Swept, confocally-aligned planar excitation (SCAPE) microscopy is a 3D, light-sheet imaging technique capable of acquiring large fields of view with high spatiotemporal resolution. While conventional light sheet microscopes utilize two or more orthogonal objectives for their excitation and detection paths, SCAPE microscopy accommodates both light paths within a single high numerical aperture objective. This geometry permits SCAPE to circumvent optomechanical limitations that constrain both sample selection and 3D imaging speed in conventional light sheet systems, while delivering similar advantages, including low phototoxicity and relatively simple instrumentation. As a result, SCAPE can deliver these benefits, and high 3D imaging speeds to application spaces more usually met by laser-scanning techniques such as confocal and two-photon microscopy.

Thus far, we have evaluated SCAPE microscopy’s ability to image a range of intact, behaving organisms including freely crawling Drosophila larvae, the whole brain of adult, behaving Drosophila, the whole larval zebrafish brain, C. Elegans worms and the awake, behaving mouse brain. With each application we have developed optimized experimental methods and system configurations to meet requirements, including adaptable fields of view, volume rates, depths of field, spatial resolution, and spectral multiplexing.

Here, we will describe the development of these second generation SCAPE systems, including a simplified design that offers much more versatility and better performance than our first-generation system. In addition to a larger, more uniform field of view with better resolution, these improvements have yielded volumetric imaging speeds up to and exceeding 100 Hz.

We will showcase the system’s ability to adapt to the unique requirements of different model organisms and present our latest neuroimaging results.

36. Voltage Imaging from Dendritic Spines Using Patterned Illumination Based on Computer-Generated Holography (CGH). JU-YUN WENG,¹ DIMITRII TANESE², VALERIA ZAMPINI,² VINCENT DE SARS², MARCO CANEPARI,^3,4,5 BALAZS ROZSA,⁶ VALENTINA EMILIANI,² and DEJAN ZECEVIC,^{1 1}Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520; ²Neurophotonics Laboratory, CNRS UMR8250, Paris Descartes University, 75270 Paris, France; ³Laboratory for Interdisciplinary Physics, UMR 5588, Université Grenoble Alpes and CNRS, 38402 Saint Martin d’Hères, France; ⁴Laboratories of Excellence, Ion Channel Science and Therapeutics, France; ⁵Institut National de la Santé et Recherche Médicale (INSERM), France; ⁶Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest H-1083, Hungary

Additional research is necessary to clarify electrical role of dendritic spines. Notwithstanding decades of theoretical and modeling studies, as well as calcium, sodium, and voltage imaging experiments and nano-electrode recordings, the elementary question of the value of spine neck resistance relative to the input impedance of thin spiny dendrites is controversial. This controversy allows for different hypotheses about electrical role played by dendritic spines. To obtained direct evidence, we previously used an electrochromic voltage-sensitive dye (JPW3028) as a transmembrane optical voltmeter with a linear scale capable of recording simultaneously electrical signals from individual spines and parent dendrites. The results showed that the spine neck does not electrically isolate synapses on spines. Thus, synapses on spine heads behave as if they are located directly on dendrites. However, scattered light and photodynamic damage currently limit the investigation of more complex electrical phenomena that require many repeated measurements from dendritic spines. Here, we demonstrate a dual-color computer generated holography (CGH) illumination system, which was used for multisite optical recording and glutamate uncaging. The specific intensity-graded illumination patterns were designed to eliminate contamination of spine signals by the scattered light from parent dendrites thereby increasing the effective spatial resolution of voltage imaging. Additionally, the restriction of the high-intensity illumination exclusively to regions of interest (spine heads and small sections on parent dendrites) can significantly reduce photodynamic damage. Furthermore, in parallel with voltage imaging, we successfully used a second CGH system for multisite glutamate uncaging. Overall, a dual-color CGH illumination system drastically improved the spatial resolution of voltage imaging, reduced photodynamic damage, and provided true simultaneous multiple site uncaging of glutamate.

37. Developing an Optogenetic Tool for Tethering Mitochondria and Endoplasmic Reticulum in Neurons. MASAYUKI YAZAWA and FAN SHI, Department of Rehabilitation and Regenerative Medicine, Columbia Stem Cell Initiative, Columbia University, New York

Interaction between mitochondria and endoplasmic reticulum (ER) is essential for calcium handling, metabolism, and autophagy. Here, we present novel light-induced systems to control the interaction between mitochondria and ER in mammalian cell lines and primary neurons. To develop this system, first we have tested and optimized multiple light-induced heterodimer domains in mammalian cell lines, HEK 293T and NIH 3T3 cells. Using electron and confocal microscopes with the optimized systems, next we confirmed that blue light (∼470 nm, shot illumination ∼1 sec) could induce tethering of ER with mitochondria immediately. In addition, we developed reversible and non-reversible types of light-induced systems for tethering mitochondria and ER for further applications in primary neurons. This new optogenetic tool will help us study the molecular mechanisms underlying neuronal calcium signaling, metabolism, and autophagy.

38. Synaptic Targeting Genetically Encoded Voltage Indicators. SHENG ZHONG, DOUGLAS STORACE, and LAWRENCE B.COHEN, Dept. of Cellular and Molecular Physiology, Yale University School of Medicine

Genetically encoded voltage indicators (GEVIs) offer direct measurement of neuron membrane potential changes with fluorescence changes. While the molecular mechanism of GEVIs were explored (Storace et al. 2016. Trends Neurosci. 39(5):277–289), further improvements on spatial and temporal resolution are still highly desired. GEVIs can express well on neuronal cell membrane, but the fluorescence in unwanted region would raise up background noise. For more accurate measurement, we generate synaptic targeting constructs of Arclight for mammalian neuron and zebrafish neurons, respectively, with postsynaptic-interacting protein. The synaptic targeting GEVI can give quantal resolution imaging of synaptic transmission in cultured cells and transgenic animals that can discover short-term synaptic plasticity and locomotor behavior.