A hypothesis and the experiments to test it propose that very long-term memories, such as fear conditioning, are stored as the pattern of holes in the perineuronal net (PNN), a specialized ECM that ...envelops mature neurons and restricts synapse formation. The 3D intertwining of PNN and synapses would be imaged by serial-section EM. Lifetimes of PNN vs. intrasynaptic components would be compared with pulse-chase ¹⁵N labeling in mice and ¹⁴C content in human cadaver brains. Genetically encoded indicators and antineoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. Further techniques suggested include genetic KOs, better pharmacological inhibitors, and a genetically encoded snapshot reporter, which will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, drive expression of effector genes to mark those cells, and allow selective excitation, inhibition, or ablation to test their functional importance. The snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Finally, biosynthesis of PNN components and proteases would be imaged.
Trip the light fantastic: The green fluorescent protein (GFP) is an invaluable tool for biochemical and medicinal research. It can make tumors, amyloid plaques from Alzheimer′s disease, or pathogenic ...bacteria equally visible. Ground‐breaking contributions in this field have resulted in the 2008 Nobel Prize for Chemistry being awarded to Osamu Shimomura, Martin Chalfie, and Roger Tsien. The Nobel Laureates describe their research first‐hand.
Channelrhodopsins (ChRs) are used to optogenetically depolarize neurons. We engineered a variant of ChR, denoted red-activatable ChR (ReaChR), that is optimally excited with orange to red light (λ ...∼590-630 nm) and offers improved membrane trafficking, higher photocurrents and faster kinetics compared to existing red-shifted ChRs. Red light is less scattered by tissue and is absorbed less by blood than the blue to green wavelengths that are required by other ChR variants. We used ReaChR expressed in the vibrissa motor cortex to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illumination with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window or implant optical fibers.
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ ...visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.
Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic ...range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Monomeric red and far-red FPs and indicators now perform nearly as well as the best green FPs (and indicators).
Reversible and irreversible photochromism in FPs can be exploited to increase optical resolution and improve contrast compared with traditional fluorescence microscopy.
Infrared FPs (IFPs) are becoming ever more useful as labels for various proteins that allow correct localization and whole-animal imaging. IFPs can serve as an additional fluorescent ‘color’ for simultaneous imaging with visible FP-labeled proteins.
Bacterial phytochrome (BphP)-based IFPs provide a new scaffold for engineering fluorogenic indicators, which are ideal to visualize spatiotemporal dynamics of cell signaling in vivo.
Small ultra-red FP (smURFP) is the brightest far-red nonprototypical FP (comparable with EGFP) and is extremely photostable. smURFP may prove particularly useful as a photostable FP for super-resolution imaging and as a FRET acceptor for biosensing applications.
The engineering of new fluorescent indicators that combine features of prototypical FP-based indicators with photochromic proteins can reveal the cellular maps of biochemical activities in super-resolution.
FPs can be used as optogenetic actuators to manipulate cellular and protein functions through chromophore-assisted light inactivation or light-controlled protein oligomerization.
A glowing new era in cancer surgery may be dawning. Using fluorescently labelled markers, surgical molecular navigation means that tumours and nerves can be displayed in real time intra-operatively ...in contrasting pseudocolours, which allows more complete tumour resection while preserving important structures. These advances can potentially cause a paradigm shift in cancer surgery, improving patient outcome and decreasing overall health-care costs.
Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by ...poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M⁻¹ cm⁻¹; and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.
Optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. In the current study, we engineered an optogenetic technique that directly inhibits ...neurotransmitter release. We used a genetically encoded singlet oxygen generator, miniSOG, to conduct chromophore assisted light inactivation (CALI) of synaptic proteins. Fusions of miniSOG to VAMP2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. In cultured neurons and hippocampal organotypic slices, synaptic release was reduced up to 100%. Such inhibition lasted >1 hr and had minimal effects on membrane electrical properties. When miniSOG-VAMP2 was expressed panneuronally in Caenorhabditis elegans, movement of the worms was reduced after illumination, and paralysis was often observed. The movement of the worms recovered overnight. We name this technique Inhibition of Synapses with CALI (InSynC). InSynC is a powerful way to silence genetically specified synapses with light in a spatially and temporally precise manner.
•MiniSOG mediates CALI of presynaptic release proteins•Synaptic release is inhibited optogenetically with miniSOG fused VAMP2 or SYP1•Light reversibly reduces movement in miniSOG-VAMP2-expressing C. elegans
Lin et al. tether miniSOG to presynaptic proteins allowing optogenetic inhibition of synaptic release. This technique, InSynC, effectively inhibits synapses in cultured neurons, organotypic slice, and behaving C. elegans and is useful for studying the function of specific projections.
Engineering a memory with LTD and LTP Nabavi, Sadegh; Fox, Rocky; Proulx, Christophe D ...
Nature (London),
07/2014, Letnik:
511, Številka:
7509
Journal Article
Recenzirano
Odprti dostop
It has been proposed that memories are encoded by modification of synaptic strengths through cellular mechanisms such as long-term potentiation (LTP) and long-term depression (LTD). However, the ...causal link between these synaptic processes and memory has been difficult to demonstrate. Here we show that fear conditioning, a type of associative memory, can be inactivated and reactivated by LTD and LTP, respectively. We began by conditioning an animal to associate a foot shock with optogenetic stimulation of auditory inputs targeting the amygdala, a brain region known to be essential for fear conditioning. Subsequent optogenetic delivery of LTD conditioning to the auditory input inactivates memory of the shock. Then subsequent optogenetic delivery of LTP conditioning to the auditory input reactivates memory of the shock. Thus, we have engineered inactivation and reactivation of a memory using LTD and LTP, supporting a causal link between these synaptic processes and memory.
Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a ...recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.
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