Two-dimensional electro-optical multiphoton microscopy.

Significance: The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging in vivo. Aim: To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast in vivo imaging of genetically encoded indicators with millisecond dynamics. Approach: The performance of a commercially available KTN-EOD was characterized across a range of drive frequencies and laser parameters relevant to in vivo 2P microscopy. A second KTN-EOD was incorporated into a dual-axis scan module, and the system was validated by imaging signals in vivo from ASAP3, a genetically encoded voltage indicator. Results: Optimal KTN-EOD deflection of laser light with a central wavelength of 960 nm was obtained up to the highest average powers and pulse intensities tested (power: 350 mW; pulse duration: 118 fs). Up to 32 resolvable spots per line at a 560 kHz line scan rate could be obtained with single-axis deflection. The complete dual-axis EO 2P microscope was capable of imaging a 13 µm by 13 µm field-of-view at over 10 kHz frame rate with ∼0.5 µm lateral resolution. We demonstrate in vivo imaging of neurons expressing ASAP3 with high temporal resolution. Conclusions: We demonstrate the suitability of KTN-EODs for ultrafast 2P cellular imaging in vivo, providing a foundation for future high-performance microscopes to incorporate emerging advances in KTN-based scanning technology.

Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging.

Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (Pr). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets Pr remain to be defined. To address this, we developed QuaSOR, a super-resolution method for determining Pr from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. We mapped the Pr onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). We find that Pr varies greatly between synapses made by a single axon, quantify the contribution of key AZ proteins to Pr diversity and find that one of these, Complexin, suppresses spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission.

High-performance, compact optical standard.

We describe a high-performance, compact optical frequency standard based on a microfabricated Rb vapor cell and a low-noise, external cavity diode laser operating on the Rb two-photon transition at 778 nm. The optical standard achieves an instability of 1.8×10-13τ-1/2 for times less than 100 s and a flicker noise floor of 1×10-14 out to 6000 s. At long integration times, the instability is limited by variations in optical probe power and the ac Stark shift. The retrace was measured to 5.7×10-13 after 30 h of dormancy. Such a simple, yet high-performance optical standard could be suitable as an accurate realization of the meter or, if coupled with an optical frequency comb, as a compact atomic clock comparable to a hydrogen maser.

Miniaturized optical frequency reference for next-generation portable optical clocks.

Optical frequency standards, or lasers stabilized to atomic or molecular transitions, are widely used in length metrology and laser ranging, provide a backbone for optical communications and lie at the heart of next-generation optical atomic clocks. Here we demonstrate a compact, low-power optical frequency reference based on the Doppler-free, two-photon transition in rubidium-87 at 778 nm implemented on a micro-optics breadboard. Our optical reference achieves a fractional frequency instability of 2.9×10-12/τ for averaging times τ less than 103 s, has a volume of ≈35 cm3 and operates on ≈450 mW of electrical power. The advanced optical integration presented here demonstrates a key step towards the development of compact optical clocks and the broad dissemination of SI-traceable wavelength references.

Nanophotonic tantala waveguides for supercontinuum generation pumped at 1560 nm.

We experimentally demonstrate efficient and broadband supercontinuum generation in nonlinear tantala (Ta2O5) waveguides using a 1560 nm femtosecond seed laser. With incident pulse energies as low as 100 pJ, we create spectra spanning up to 1.6 octaves across the visible and infrared. Fabricated devices feature propagation losses as low as 10 dB/m, and they can be dispersion engineered through lithographic patterning for specific applications. We show a waveguide design suitable for low-power self-referencing of a fiber frequency comb that produces dispersive-wave radiation directly at the second-harmonic wavelength of the seed laser. A fiber-connectorized, hermetically sealed module with 2 dB per facet insertion loss and watt-level average-power handling is also described. Highly efficient and fully packaged tantala waveguides may open new possibilities for the integration of nonlinear nanophotonics into systems for precision timing, quantum science, biological imaging, and remote sensing.

Carrier of Wingless (Cow) Regulation of Drosophila Neuromuscular Junction Development.

The first Wnt signaling ligand discovered, Drosophila Wingless [Wg (Wnt1 in mammals)], plays critical roles in neuromuscular junction (NMJ) development, regulating synaptic architecture, and function. Heparan sulfate proteoglycans (HSPGs), consisting of a core protein with heparan sulfate (HS) glycosaminoglycan (GAG) chains, bind to Wg ligands to control both extracellular distribution and intercellular signaling function. Drosophila HSPGs previously shown to regulate Wg trans-synaptic signaling at the NMJ include the glypican Dally-like protein (Dlp) and perlecan Terribly Reduced Optic Lobes (Trol). Here, we investigate synaptogenic functions of the most recently described Drosophila HSPG, secreted Carrier of Wingless (Cow), which directly binds Wg in the extracellular space. At the glutamatergic NMJ, we find that Cow secreted from the presynaptic motor neuron acts to limit synaptic architecture and neurotransmission strength. In cow null mutants, we find increased synaptic bouton number and elevated excitatory current amplitudes, phenocopying presynaptic Wg overexpression. We show cow null mutants exhibit an increased number of glutamatergic synapses and increased synaptic vesicle fusion frequency based both on GCaMP imaging and electrophysiology recording. We find that membrane-tethered Wg prevents cow null defects in NMJ development, indicating that Cow mediates secreted Wg signaling. It was shown previously that the secreted Wg deacylase Notum restricts Wg signaling at the NMJ, and we show here that Cow and Notum work through the same pathway to limit synaptic development. We conclude Cow acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix.

Publisher Correction: Oblique-plane single-molecule localization microscopy for tissues and small intact animals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Oblique-plane single-molecule localization microscopy for tissues and small intact animals.

Single-molecule localization microscopy (SMLM), while well established for cultured cells, is not yet fully compatible with tissue-scale samples. We introduce single-molecule oblique-plane microscopy (obSTORM), which by directly imaging oblique sections of samples with oblique light-sheet illumination offers a deep and volumetric SMLM platform that is convenient for standard tissue samples and small intact animals. We demonstrate super-resolution imaging at depths of up to 66 µm for cells, Caenorhabditis elegans gonads, Drosophila melanogaster larval brain, mouse retina and brain sections, and whole stickleback fish.

Input-Specific Plasticity and Homeostasis at the Drosophila Larval Neuromuscular Junction.

Synaptic connections undergo activity-dependent plasticity during development and learning, as well as homeostatic re-adjustment to ensure stability. Little is known about the relationship between these processes, particularly in vivo. We addressed this with novel quantal resolution imaging of transmission during locomotive behavior at glutamatergic synapses of the Drosophila larval neuromuscular junction. We find that two motor input types, Ib and Is, provide distinct forms of excitatory drive during crawling and differ in key transmission properties. Although both inputs vary in transmission probability, active Is synapses are more reliable. High-frequency firing "wakes up" silent Ib synapses and depresses Is synapses. Strikingly, homeostatic compensation in presynaptic strength only occurs at Ib synapses. This specialization is associated with distinct regulation of postsynaptic CaMKII. Thus, basal synaptic strength, short-term plasticity, and homeostasis are determined input-specifically, generating a functional diversity that sculpts excitatory transmission and behavioral function.

Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging.

Circuit mapping requires knowledge of both structural and functional connectivity between cells. Although optical tools have been made to assess either the morphology and projections of neurons or their activity and functional connections, few probes integrate this information. We have generated a family of photoactivatable genetically encoded Ca(2+) indicators that combines attributes of high-contrast photolabeling with high-sensitivity Ca(2+) detection in a single-color protein sensor. We demonstrated in cultured neurons and in fruit fly and zebrafish larvae how single cells could be selected out of dense populations for visualization of morphology and high signal-to-noise measurements of activity, synaptic transmission and connectivity. Our design strategy is transferrable to other sensors based on circularly permutated GFP (cpGFP).

Evoked and spontaneous transmission favored by distinct sets of synapses.

BACKGROUND: Spontaneous "miniature" transmitter release takes place at low rates at all synapses. Long thought of as an unavoidable leak, spontaneous release has recently been suggested to be mediated by distinct pre- and postsynaptic molecular machineries and to have a specialized role in setting up and adjusting neuronal circuits. It remains unclear how spontaneous and evoked transmission are related at individual synapses, how they are distributed spatially when an axon makes multiple contacts with a target, and whether they are commonly regulated. RESULTS: Electrophysiological recordings in the Drosophila larval neuromuscular junction, in the presence of the use-dependent glutamate receptor (GluR) blocker philanthotoxin, indicated that spontaneous and evoked transmission employ distinct sets of GluRs. In vivo imaging of transmission using synaptically targeted GCaMP3 to detect Ca(2+) influx through the GluRs revealed little spatial overlap between synapses participating in spontaneous and evoked transmission. Spontaneous and evoked transmission were oppositely correlated with presynaptic levels of the protein Brp: synapses with high Brp favored evoked transmission, whereas synapses with low Brp were more active spontaneously. High-frequency stimulation did not increase the overlap between evoked and spontaneous transmission, and instead decreased the rate of spontaneous release from synapses that were highly active in evoked transmission. CONCLUSIONS: Although individual synapses can participate in both evoked and spontaneous transmission, highly active synapses show a preference for one mode of transmission. The presynaptic protein Brp promotes evoked transmission and suppresses spontaneous release. These findings suggest the existence of presynaptic mechanisms that promote synaptic specialization to either evoked or spontaneous transmission.

Cyclooxygenase-2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine-induced presynaptic enhancement at the vertebrate neuromuscular junction.

Previous work has demonstrated that activation of muscarinic acetylcholine receptors at the lizard neuromuscular junction (NMJ) induces a biphasic modulation of evoked neurotransmitter release: an initial depression followed by a delayed enhancement. The depression is mediated by the release of the endocannabinoid 2-arachidonylglycerol (2-AG) from the muscle and its binding to cannabinoid type 1 receptors on the motor nerve terminal. The work presented here suggests that the delayed enhancement of neurotransmitter release is mediated by cyclooxygenase-2 (COX-2) as it converts 2-AG to the glycerol ester of prostaglandin E2 (PGE2-G). Using immunofluorescence, COX-2 was detected in the perisynaptic Schwann cells (PSCs) surrounding the NMJ. Pretreatment with either of the selective COX-2 inhibitors, nimesulide or DuP 697, prevents the delayed increase in endplate potential (EPP) amplitude normally produced by muscarine. In keeping with its putative role as a mediator of the delayed muscarinic effect, PGE2-G enhances evoked neurotransmitter release. Specifically, PGE2-G increases the amplitude of EPPs without altering that of spontaneous miniature EPPs. As shown previously for the muscarinic effect, the enhancement of evoked neurotransmitter release by PGE2-G depends on nitric oxide (NO) as the response is abolished by application of either N(G)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthesis, or carboxy-PTIO, a chelator of NO. Intriguingly, the enhancement is not prevented by AH6809, a prostaglandin receptor antagonist, but is blocked by capsazepine, a TRPV1 and TRPM8 receptor antagonist. Taken together, these results suggest that the conversion of 2-AG to PGE2-G by COX-2 underlies the muscarine-induced enhancement of neurotransmitter release at the vertebrate NMJ.

Extrasynaptic glutamate and inhibitory neurotransmission modulate ganglion cell participation during glutamatergic retinal waves.

During the first 2 wk of mouse postnatal development, transient retinal circuits give rise to the spontaneous initiation and lateral propagation of depolarizations across the ganglion cell layer (GCL). Glutamatergic retinal waves occur during the second postnatal week, when GCL depolarizations are mediated by ionotropic glutamate receptors. Bipolar cells are the primary source of glutamate in the inner retina, indicating that the propagation of waves depends on their activation. Using the fluorescence resonance energy transfer-based optical sensor of glutamate FLII81E-1µ, we found that retinal waves are accompanied by a large transient increase in extrasynaptic glutamate throughout the inner plexiform layer. Using two-photon Ca(2+) imaging to record spontaneous Ca(2+) transients in large populations of cells, we found that despite this spatially diffuse source of depolarization, only a subset of neurons in the GCL and inner nuclear layer (INL) are robustly depolarized during retinal waves. Application of the glutamate transporter blocker dl-threo-ß-benzyloxyaspartate (25 µM) led to a significant increase in cell participation in both layers, indicating that the concentration of extrasynaptic glutamate affects cell participation in both the INL and GCL. In contrast, blocking inhibitory transmission with the GABAA receptor antagonist gabazine and the glycine receptor antagonist strychnine increased cell participation in the GCL without significantly affecting the INL. These data indicate that during development, glutamate spillover provides a spatially diffuse source of depolarization, but that inhibitory circuits dictate which neurons within the GCL participate in retinal waves.

Anthrax lethal factor cleaves mouse nlrp1b in both toxin-sensitive and toxin-resistant macrophages.

Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.

Anthrax lethal factor cleavage of Nlrp1 is required for activation of the inflammasome.

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1ß and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 ß release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.

Auranofin protects against anthrax lethal toxin-induced activation of the Nlrp1b inflammasome.

Anthrax lethal toxin (LT) is the major virulence factor for Bacillus anthracis. The lethal factor (LF) component of this bipartite toxin is a protease which, when transported into the cellular cytoplasm, cleaves mitogen-activated protein kinase kinase (MEK) family proteins and induces rapid toxicity in mouse macrophages through activation of the Nlrp1b inflammasome. A high-throughput screen was performed to identify synergistic LT-inhibitory drug combinations from within a library of approved drugs and molecular probes. From this screen we discovered that auranofin, an organogold compound with anti-inflammatory activity, strongly inhibited LT-mediated toxicity in mouse macrophages. Auranofin did not inhibit toxin transport into cells or MEK cleavage but inhibited both LT-mediated caspase-1 activation and caspase-1 catalytic activity. Thus, auranofin inhibited LT-mediated toxicity by preventing activation of the Nlrp1b inflammasome and the downstream actions that occur in response to the toxin. Idebenone, an analog of coenzyme Q, synergized with auranofin to increase its protective effect. We found that idebenone functions as an inhibitor of voltage-gated potassium channels and thus likely mediates synergy through inhibition of the potassium fluxes which have been shown to be required for Nlrp1b inflammasome activation.

Inflammasome sensor Nlrp1b-dependent resistance to anthrax is mediated by caspase-1, IL-1 signaling and neutrophil recruitment.

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1b(S/S) or Nlrp1b(R/R), respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1b(S/S) alleles (which allow activation of caspase-1 and IL-1ß release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1b(R/R) alleles (which cannot activate caspase-1 in response to toxin). Nlrp1b(S)-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1b(S/S) mice. Resistance to infection required the actions of both caspase-1 and IL-1ß as Nlrp1b(S/S) mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1ß responses in Nlrp1b(S/S),Nlrp1b(R/) (R) and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1b(S), caspase-1, and IL-1ß in countering anthrax infection.

Anthrax lethal toxin activates the inflammasome in sensitive rat macrophages.

Anthrax lethal toxin (LT) is an important virulence factor for Bacillus anthracis. In mice, LT lyses macrophages from certain inbred strains in less than 2h by activating the Nlrp1b inflammasome and caspase-1, while macrophages from other strains remain resistant to the toxin's effects. We analyzed LT effects in toxin-sensitive and resistant rat macrophages to test if a similar pathway was involved in rat macrophage death. LT activates caspase-1 in rat macrophages from strains harboring LT-sensitive macrophages in a manner similar to that in toxin-sensitive murine macrophages. This activation of caspase-1 is dependent on proteasome activity, and sensitive macrophages are protected from LT's lytic effects by lactacystin. Proteasome inhibition also delayed the death of rats in response to LT, confirming our previous data implicating the rat Nlrp1 inflammasome in animal death. Quinidine, caspase-1 inhibitors, the cathepsin B inhibitor CA-074Me, and heat shock also protected rat macrophages from LT toxicity. These data support the existence of an active functioning LT-responsive Nlrp1 inflammasome in rat macrophages. The activation of the rat Nlrp1 inflammasome is required for LT-mediated rat macrophage lysis and contributes to animal death.

Susceptibility to anthrax lethal toxin-induced rat death is controlled by a single chromosome 10 locus that includes rNlrp1.

Anthrax lethal toxin (LT) is a bipartite protease-containing toxin and a key virulence determinant of Bacillus anthracis. In mice, LT causes the rapid lysis of macrophages isolated from certain inbred strains, but the correlation between murine macrophage sensitivity and mouse strain susceptibility to toxin challenge is poor. In rats, LT induces a rapid death in as little as 37 minutes through unknown mechanisms. We used a recombinant inbred (RI) rat panel of 19 strains generated from LT-sensitive and LT-resistant progenitors to map LT sensitivity in rats to a locus on chromosome 10 that includes the inflammasome NOD-like receptor (NLR) sensor, Nlrp1. This gene is the closest rat homolog of mouse Nlrp1b, which was previously shown to control murine macrophage sensitivity to LT. An absolute correlation between in vitro macrophage sensitivity to LT-induced lysis and animal susceptibility to the toxin was found for the 19 RI strains and 12 additional rat strains. Sequencing Nlrp1 from these strains identified five polymorphic alleles. Polymorphisms within the N-terminal 100 amino acids of the Nlrp1 protein were perfectly correlated with LT sensitivity. These data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1.

CA-074Me protection against anthrax lethal toxin.

Anthrax lethal toxin (LT) activates the NLRP1b (NALP1b) inflammasome and caspase-1 in macrophages from certain inbred mouse strains, but the mechanism by which this occurs is poorly understood. We report here that similar to several NLRP3 (NALP3, cryopyrin)-activating stimuli, LT activation of the NLRP1b inflammasome involves lysosomal membrane permeabilization (LMP) and subsequent cytoplasmic cathepsin B activity. CA-074Me, a potent cathepsin B inhibitor, protects LT-sensitive macrophages from cell death and prevents the activation of caspase-1. RNA interference knockdown of cathepsin B expression, however, cannot prevent LT-mediated cell death, suggesting that CA-074Me may also act on other cellular proteases released during LMP. CA-074Me appears to function downstream of LT translocation to the cytosol (as assessed by mitogen-activated protein kinase kinase cleavage), K(+) effluxes, and proteasome activity. The initial increase in cytoplasmic activity of cathepsin B occurs at the same time or shortly before caspase-1 activation but precedes a larger-scale lysosomal destabilization correlated closely with cytolysis. We present results suggesting that LMP may be involved in the activation of the NLRP1b inflammasome.