Regional synapse gain and loss accompany memory formation in larval zebrafish.

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670-675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604-607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull 57, 397-399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

Structure and Function of an Actin-Based Filter in the Proximal Axon.

The essential organization of microtubules within neurons has been described; however, less is known about how neuronal actin is arranged and the functional implications of its arrangement. Here, we describe, in live cells, an actin-based structure in the proximal axon that selectively prevents some proteins from entering the axon while allowing the passage of others. Concentrated patches of actin in proximal axons are present shortly after axonal specification in rat and zebrafish neurons imaged live, and they mark positions where anterogradely traveling vesicles carrying dendritic proteins halt and reverse. Patches colocalize with the ARP2/3 complex, and when ARP2/3-mediated nucleation is blocked, a dendritic protein mislocalizes to the axon. Patches are highly dynamic, with few persisting longer than 30 min. In neurons in culture and in vivo, actin appears to form a contiguous, semipermeable barrier, despite its apparently sparse distribution, preventing axonal localization of constitutively active myosin Va but not myosin VI.

Adaptive optics improves multiphoton super-resolution imaging.

We improve multiphoton structured illumination microscopy using a nonlinear guide star to determine optical aberrations and a deformable mirror to correct them. We demonstrate our method on bead phantoms, cells in collagen gels, nematode larvae and embryos, Drosophila brain, and zebrafish embryos. Peak intensity is increased (up to 40-fold) and resolution recovered (up to 176 ± 10 nm laterally, 729 ± 39 nm axially) at depths ∼250 µm from the coverslip surface.

An E3-ligase-based method for ablating inhibitory synapses.

Although neuronal activity can be modulated using a variety of techniques, there are currently few methods for controlling neuronal connectivity. We introduce a tool (GFE3) that mediates the fast, specific and reversible elimination of inhibitory synaptic inputs onto genetically determined neurons. GFE3 is a fusion between an E3 ligase, which mediates the ubiquitination and rapid degradation of proteins, and a recombinant, antibody-like protein (FingR) that binds to gephyrin. Expression of GFE3 leads to a strong and specific reduction of gephyrin in culture or in vivo and to a substantial decrease in phasic inhibition onto cells that express GFE3. By temporarily expressing GFE3 we showed that inhibitory synapses regrow following ablation. Thus, we have created a simple, reversible method for modulating inhibitory synaptic input onto genetically determined cells.

Determination of the source of SHG verniers in zebrafish skeletal muscle.

SHG microscopy is an emerging microscopic technique for medically relevant imaging because certain endogenous proteins, such as muscle myosin lattices within muscle cells, are sufficiently spatially ordered to generate detectable SHG without the use of any fluorescent dye. Given that SHG signal is sensitive to the structural state of muscle sarcomeres, SHG functional imaging can give insight into the integrity of muscle cells in vivo. Here, we report a thorough theoretical and experimental characterization of myosin-derived SHG intensity profiles within intact zebrafish skeletal muscle. We determined that "SHG vernier" patterns, regions of bifurcated SHG intensity, are illusory when sarcomeres are staggered with respect to one another. These optical artifacts arise due to the phase coherence of SHG signal generation and the Guoy phase shift of the laser at the focus. In contrast, two-photon excited fluorescence images obtained from fluorescently labeled sarcomeric components do not contain such illusory structures, regardless of the orientation of adjacent myofibers. Based on our results, we assert that complex optical artifacts such as SHG verniers should be taken into account when applying functional SHG imaging as a diagnostic readout for pathological muscle conditions.

In vivo single-cell labeling by confined primed conversion.

Spatially confined green-to-red photoconversion of fluorescent proteins with high-power, pulsed laser illumination is negligible, thus precluding optical selection of single cells in vivo. We report primed conversion, in which low-power, dual-wavelength, continuous-wave illumination results in pronounced photoconversion. With a straightforward addition to a conventional confocal microscope, we show confined primed conversion in living zebrafish and reveal the complex anatomy of individual neurons packed between neighboring cells.

In vivo cell tracking using PhOTO zebrafish.

By combining the strength of previously described in vivo cell tracking methodologies, we have recently generated a set of transgenic zebrafish lines, called "PhOTO (photoconvertible optical tracking of…)" zebrafish. PhOTO zebrafish lines are suitable for cell tracking during highly dynamic events, including gastrulation, tissue regeneration, tumorigenesis, and cancer/disease progression. Global monitoring of cell shape, cell interactions, e.g., cell intercalations, coordinated division, and cell dynamics are accomplished by using fluorescence imaging of nuclear and plasma membrane fluorescent protein labeling. The irreversible green-to-red photoconversion property of Dendra2 fusions enables noninvasive, specific and high-contrast selection of targeted cells of interest, which greatly simplifies cell tracking and segmentation in time and space. Here we demonstrate photoconversion and in vivo cell tracking using PhOTO zebrafish.

Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish.

To address the need for a bright, photostable labeling tool that allows long-term in vivo imaging in whole organisms, we recently introduced second harmonic generating (SHG) nanoprobes. Here we present a protocol for the preparation and use of a particular SHG nanoprobe label, barium titanate (BT), for in vivo imaging in living zebrafish embryos. Chemical treatment of the BT nanoparticles results in surface coating with amine-terminal groups, which act as a platform for a variety of chemical modifications for biological applications. Here we describe cross-linking of BT to a biotin-linked moiety using click chemistry methods and coating of BT with nonreactive poly(ethylene glycol) (PEG). We also provide details for injecting PEG-coated SHG nanoprobes into zygote-stage zebrafish embryos, and in vivo imaging of SHG nanoprobes during gastrulation and segmentation. Implementing the PROCEDURE requires a basic understanding of laser-scanning microscopy, experience with handling zebrafish embryos and chemistry laboratory experience. Functionalization of the SHG nanoprobes takes ∼3 d, whereas zebrafish preparation, injection and imaging setup should take approximately 2-4 h.

PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration.

BACKGROUND: Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo. METHODOLOGY: PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100 µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin. CONCLUSIONS/SIGNIFICANCE: PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior.

SHG nanoprobes: advancing harmonic imaging in biology.

Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination - converting two photons into one photon of half the incident wavelength - and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution.

Probabilistic sensitivity analysis of biochemical reaction systems.

Sensitivity analysis is an indispensable tool for studying the robustness and fragility properties of biochemical reaction systems as well as for designing optimal approaches for selective perturbation and intervention. Deterministic sensitivity analysis techniques, using derivatives of the system response, have been extensively used in the literature. However, these techniques suffer from several drawbacks, which must be carefully considered before using them in problems of systems biology. We develop here a probabilistic approach to sensitivity analysis of biochemical reaction systems. The proposed technique employs a biophysically derived model for parameter fluctuations and, by using a recently suggested variance-based approach to sensitivity analysis [Saltelli et al., Chem. Rev. (Washington, D.C.) 105, 2811 (2005)], it leads to a powerful sensitivity analysis methodology for biochemical reaction systems. The approach presented in this paper addresses many problems associated with derivative-based sensitivity analysis techniques. Most importantly, it produces thermodynamically consistent sensitivity analysis results, can easily accommodate appreciable parameter variations, and allows for systematic investigation of high-order interaction effects. By employing a computational model of the mitogen-activated protein kinase signaling cascade, we demonstrate that our approach is well suited for sensitivity analysis of biochemical reaction systems and can produce a wealth of information about the sensitivity properties of such systems. The price to be paid, however, is a substantial increase in computational complexity over derivative-based techniques, which must be effectively addressed in order to make the proposed approach to sensitivity analysis more practical.