Advanced deep-tissue imaging and manipulation enabled by biliverdin reductase knockout.

We developed near-infrared (NIR) photoacoustic and fluorescence probes, as well as optogenetic tools from bacteriophytochromes, and enhanced their performance using biliverdin reductase-A knock-out model (Blvra-/-). Blvra-/- elevates endogenous heme-derived biliverdin chromophore for bacteriophytochrome-derived NIR constructs. Consequently, light-controlled transcription with IsPadC-based optogenetic tool improved up to 25-fold compared to wild-type cells, with 100-fold activation in Blvra-/- neurons. In vivo , light-induced insulin production in Blvra-/- reduced blood glucose in diabetes by ∼60%, indicating high potential for optogenetic therapy. Using 3D photoacoustic, ultrasound, and two-photon fluorescence imaging, we overcame depth limitations of recording NIR probes. We achieved simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of blood vessels ∼7 mm deep in the brain, with intact scalp and skull. Two-photon microscopy provided cell-level resolution of miRFP720-expressing neurons ∼2.2 mm deep. Blvra-/- significantly enhances efficacy of biliverdin-dependent NIR systems, making it promising platform for interrogation and manipulation of biological processes.

iLight2: A near-infrared optogenetic tool for gene transcription with low background activation.

Optogenetic tools (OTs) operating in the far-red and near-infrared (NIR) region offer advantages for light-controlling biological processes in deep tissues and spectral multiplexing with fluorescent probes and OTs acting in the visible range. However, many NIR OTs suffer from background activation in darkness. Through shortening linkers, we engineered a novel NIR OT, iLight2, which exhibits a significantly reduced background activity in darkness, thereby increasing the light-to-dark activation contrast. The resultant optimal configuration of iLight2 components suggests a molecular mechanism of iLight2 action. Using a biliverdin reductase knock-out mouse model, we show that iLight2 exhibits advanced performance in mouse primary cells and deep tissues in vivo. Efficient light-controlled cell migration in wound healing cellular model demonstrates the possibility of using iLight2 in therapy and, overall, positions it as a valuable addition to the NIR OT toolkit for gene transcription applications.

On the Importance of Low-Frequency Signals in Functional and Molecular Photoacoustic Computed Tomography.

In photoacoustic computed tomography (PACT) with short-pulsed laser excitation, wideband acoustic signals are generated in biological tissues with frequencies related to the effective shapes and sizes of the optically absorbing targets. Low-frequency photoacoustic signal components correspond to slowly varying spatial features and are often omitted during imaging due to the limited detection bandwidth of the ultrasound transducer, or during image reconstruction as undesired background that degrades image contrast. Here we demonstrate that low-frequency photoacoustic signals, in fact, contain functional and molecular information, and can be used to enhance structural visibility, improve quantitative accuracy, and reduce spare-sampling artifacts. We provide an in-depth theoretical analysis of low-frequency signals in PACT, and experimentally evaluate their impact on several representative PACT applications, such as mapping temperature in photothermal treatment, measuring blood oxygenation in a hypoxia challenge, and detecting photoswitchable molecular probes in deep organs. Our results strongly suggest that low-frequency signals are important for functional and molecular PACT.

On the importance of low-frequency signals in functional and molecular photoacoustic computed tomography.

In photoacoustic computed tomography (PACT) with short-pulsed laser excitation, wideband acoustic signals are generated in biological tissues with frequencies related to the effective shapes and sizes of the optically absorbing targets. Low-frequency photoacoustic signal components correspond to slowly varying spatial features and are often omitted during imaging due to the limited detection bandwidth of the ultrasound transducer, or during image reconstruction as undesired background that degrades image contrast. Here we demonstrate that low-frequency photoacoustic signals, in fact, contain functional and molecular information, and can be used to enhance structural visibility, improve quantitative accuracy, and reduce spare-sampling artifacts. We provide an in-depth theoretical analysis of low-frequency signals in PACT, and experimentally evaluate their impact on several representative PACT applications, such as mapping temperature in photothermal treatment, measuring blood oxygenation in a hypoxia challenge, and detecting photoswitchable molecular probes in deep organs. Our results strongly suggest that low-frequency signals are important for functional and molecular PACT.

Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium Indicator.

Near-infrared (NIR) genetically encoded calcium indicators (GECIs) are becoming powerful tools for neuroscience. Because of their spectral characteristics, the use of NIR GECIs helps to avoid signal loss from the absorption by body pigments, light-scattering, and autofluorescence in mammalian tissues. In addition, NIR GECIs do not suffer from cross-excitation artifacts when used with common fluorescent indicators and optogenetics actuators. Although several NIR GECIs have been developed, there is no NIR GECI currently available that would combine the high brightness in cells and photostability with small size and fast response kinetics. Here, we report a small FRET-based NIR fluorescent calcium indicator iGECInano. We characterize iGECInano in vitro, in non-neuronal mammalian cells, and primary mouse neurons. iGECInano demonstrates the improvement in the signal-to-noise ratio and response kinetics compared to other NIR GECIs.

Transgenic mice encoding modern imaging probes: Properties and applications.

Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Optogenetic manipulation and photoacoustic imaging using a near-infrared transgenic mouse model.

Optogenetic manipulation and optical imaging in the near-infrared range allow non-invasive light-control and readout of cellular and organismal processes in deep tissues in vivo. Here, we exploit the advantages of Rhodopseudomonas palustris BphP1 bacterial phytochrome, which incorporates biliverdin chromophore and reversibly photoswitches between the ground (740-800 nm) and activated (620-680 nm) states, to generate a loxP-BphP1 transgenic mouse model. The mouse enables Cre-dependent temporal and spatial targeting of BphP1 expression in vivo. We validate the optogenetic performance of endogenous BphP1, which in the activated state binds its engineered protein partner QPAS1, to trigger gene transcription in primary cells and living mice. We demonstrate photoacoustic tomography of BphP1 expression in different organs, developing embryos, virus-infected tissues and regenerating livers, with the centimeter penetration depth. The transgenic mouse model provides opportunities for both near-infrared optogenetics and photoacoustic imaging in vivo and serves as a source of primary cells and tissues with genomically encoded BphP1.

Targeting hippocampal amyloidogenesis with SV2A protein modulator levetiracetam.

Cerebral amyloid ß (Aß) proteostasis is compromised under neuronal overexcitation, long-term neuroinflammation and brain aging. Using the animal model of LPS-induced neuroinflammation we demonstrated that treatment with levetiracetam, a specific modulator of synaptic vesicle glycoprotein SV2A, rescues abnormal synaptic vesicle (SV) fusion and neurotransmitter release, decreasing elevated hippocampal APP levels in vivo. Therapy with levetiracetam upregulates the SV2A in hippocampus and restores the level of apolipoprotein E, involved in brain Aß aggregation/clearance and resolution of inflammation. We demonstrated that oligomers of Aß1-42 and Aß1-40 peptides promote SV clustering, which reduces the rate and plateau level of subsequent homo- and heterotypic SNARE-mediated SV fusion. Oligomeric Aß1-42 lowered ΔpH gradient across the vesicular membrane, thus affecting their neurotransmitter storage capacity. In contrast, monomers of Aß1-42 and Aß1-40 had negligible impact on studied processes. Our data suggests that in the course of progression of neuroinflammation oligomeric forms of Aß1-42 and Aß1-40 can compromise the SV fusion machinery and that antiepileptic agent levetiracetam, acting on SV recycling and restricting overexcitation, is able to affect APP processing and Aß generation within the hippocampus in vivo.

Neuroprotective and Immunomodulatory Action of the Endocannabinoid System under Neuroinflammation.

Endocannabinoids (eCBs) are lipid-based retrograde messengers with a relatively short half-life that are produced endogenously and, upon binding to the primary cannabinoid receptors CB1/2, mediate multiple mechanisms of intercellular communication within the body. Endocannabinoid signaling is implicated in brain development, memory formation, learning, mood, anxiety, depression, feeding behavior, analgesia, and drug addiction. It is now recognized that the endocannabinoid system mediates not only neuronal communications but also governs the crosstalk between neurons, glia, and immune cells, and thus represents an important player within the neuroimmune interface. Generation of primary endocannabinoids is accompanied by the production of their congeners, the N-acylethanolamines (NAEs), which together with N-acylneurotransmitters, lipoamino acids and primary fatty acid amides comprise expanded endocannabinoid/endovanilloid signaling systems. Most of these compounds do not bind CB1/2, but signal via several other pathways involving the transient receptor potential cation channel subfamily V member 1 (TRPV1), peroxisome proliferator-activated receptor (PPAR)-α and non-cannabinoid G-protein coupled receptors (GPRs) to mediate anti-inflammatory, immunomodulatory and neuroprotective activities. In vivo generation of the cannabinoid compounds is triggered by physiological and pathological stimuli and, specifically in the brain, mediates fine regulation of synaptic strength, neuroprotection, and resolution of neuroinflammation. Here, we review the role of the endocannabinoid system in intrinsic neuroprotective mechanisms and its therapeutic potential for the treatment of neuroinflammation and associated synaptopathy.

Vitamin D deficiency induces the excitation/inhibition brain imbalance and the proinflammatory shift.

Vitamin D3 is among the major neurosteroids whose role in developing and adult brain is intensively studied now. Its active form 1,25(OH)2D3 regulates the expression and functioning of a range of brain-specific proteins, which orchestrate the neurotransmitter turnover, neurogenesis and neuroplasticity. Despite numerous studies of the vitamin D role in normal and pathological brain function, there is little evidence on the mechanisms of alterations in excitatory and inhibitory neurotransmission under vitamin D deficiency (VDD). Using the animal model we characterized the dysfunction of excitatory and inhibitory neurotransmission under alimentary VDD. The shift between unstimulated and evoked GABA release under VDD was largely reversed after treatment of VDD, whereas the impairments in glutamatergic system were only partially recovered after 1-month vitamin D3 supplementation. The increase of the external glutamate level and unstimulated GABA release in brain nerve terminals was associated with intensified ROS production and higher [Ca2+]i in presynapse. The negative allosteric modulation of presynaptic mGlu7 receptors significantly enhanced exocytotic GABA release, which was decreased under VDD, thereby suggesting the neuroprotective effect of such modulation of inhibitory neurotransmission. Synaptic plasma membranes and cytosolic proteins contribute to the decreased stimulated release of neurotransmitter, by being the crucial components, whose functional state is impaired under VDD. The critical changes with synaptic vesicles occurred at the docking step of the process, whereas malfunctioning of synaptic cytosolic proteins impacted the fusion event foremost. The decreased amplitude of exocytosis was inherent for non-excitable cells as well, as evidenced by lower platelet degranulation. Our data suggest the presynaptic dysfunction and proinflammatory shift as the early events in the pathogenesis of VDD-associated disorders and provide evidences for the neuroprotective role of vitamin D3.

Stearoylethanolamide interferes with retrograde endocannabinoid signalling and supports the blood-brain barrier integrity under acute systemic inflammation.

Neuroinflammation plays a prominent role in the onset of demyelinating diseases, major depressive disorder and delayed neurodegeneration. An open question remains whether pharmacological suppression of inflammation can effectively reduce the progression of these states. Bioactive lipid mediators such as N-acylethanolamines (NAEs) have an anti-inflammatory activity and are of pharmacological interest due to their endogenous on-demand production and the existence of distinct biological targets in humans and animals. Here we demonstrate for the first time, that treatment with stearoylethanolamide (SEA), a prevailing endogenously formed NAE, is neuroprotective against LPS-induced neuroinflammation in C57BL/6 male mice. SEA restricted the spreading of peripheral inflammation to the brain, and averted the activation of resident microglia and leukocyte trafficking to the brain parenchyma. Treatment with SEA per se increased the neuronal expression of cannabinoid receptors CB1/2 and brain levels of the most potent endogenous CB1/2 agonist 2-arachidonoylglycerol in vivo. SEA enhanced the amplitude of synaptic vesicle release, supported the balanced signal-to-noise ratio in glutamate- and GABAergic neurotransmission and decreased the excitotoxic risk associated with higher extracellular glutamate levels under neuroinflammation. The interference of SEA with the endocannabinoid system and presynaptic neurotransmitter release may represent an intrinsic neuroprotective mechanism that is triggered by inflammation and glutamate excitotoxicity. Thus, our data allows to consider SEA for the preventive therapy of acute and late-onset neuroinflammation-associated synaptic dysfunction and neurodegeneration.

Modulation of neurosecretion and approaches for its multistep analysis.

BACKGROUND: Neurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs. METHODS: Proposed fluorescence-based strategies and analysis of neurotransmitter transport provided the advantages in studies of exocytosis steps. We evaluated SV docking/tethering, their Ca2+-dependent fusion and release of neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in two animal models. RESULTS: Approaches enabled us to study: 1) endocytosis/Ca2+-dependent release of fluorescent carbon nanodots (CNDs) during stimulation of nerve terminals; 2) the action of levetiracetam, modulator of SV glycoprotein SV2, on fusion competence of SVs and stimulated release of GABA and glutamate; 3) impairments of several steps of neurosecretion under vitamin D3 deficiency. CONCLUSIONS: Our algorithm enabled us to verify the method validity for multidimensional analysis of SV turnover. By increasing SV docking and the size of readily releasable pool (RRP), levetiracetam is able to selectively enhance the stimulated GABA secretion in hippocampal neurons. Findings suggest that SV2 regulates RRP through impact on the number of docked/primed SVs. GENERAL SIGNIFICANCE: Methodology can be widely applied to study the stimulated neurosecretion in presynapse, regulation of SV docking, their Ca2+-dependent fusion with target membranes, quantitative analysis of expression of neuron-specific proteins, as well as for testing the efficiency of pre-selected designed neuroactive substances.

4-Аminopyridine sequesters intracellular Ca2+ which triggers exocytosis in excitable and non-excitable cells.

4-aminopyridine is commonly used to stimulate neurotransmitter release resulting from sustained plasma membrane depolarization and Ca2+-influx from the extracellular space. This paper elucidated unconventional mechanism of 4-aminopyridine-stimulated glutamate release from neurons and non-neuronal cells which proceeds in the absence of external Ca2+. In brain nerve terminals, primary neurons and platelets 4-aminopyridine induced the exocytotic release of glutamate that was independent of external Ca2+ and was triggered by the sequestration of Ca2+ from intracellular stores. The initial level of 4-aminopyridine-stimulated glutamate release from neurons in the absence or presence of external Ca2+ was subequal and the difference was predominantly associated with subsequent tonic release of glutamate in Ca2+-supplemented medium. The increase in [Ca2+]i and the secretion of glutamate stimulated by 4-aminopyridine in Ca2+-free conditions have resulted from Ca2+ efflux from endoplasmic reticulum and were abolished by intracellular free Ca2+ chelator BAPTA. This suggests that Ca2+ sequestration plays a profound role in the 4-aminopyridine-mediated stimulation of excitable and non-excitable cells. 4-Aminopyridine combines the properties of depolarizing agent with the ability to sequester intracellular Ca2+. The study unmasks additional mechanism of action of 4-aminopyridine, an active substance of drugs for treatment of multiple sclerosis and conditions related to reduced Ca2+ efflux from intracellular stores.

Anion carrier formation by calix[4]arene-bis-hydroxymethylphosphonic acid in bilayer membranes.

The action of calix[4]arenes C-91, C-97, C-99, C-107 and C-160 on solvent-containing planar bilayer membranes made of cholesterol and egg phosphatidylcholine (egg PC) or synthetic 18-carbon-tail phospholipid DOPC has been investigated in a voltage-clamp mode. Within the range of calix[4]arenes tested, a steady-state voltage-dependent transmembrane current was achieved only after addition of calix[4]-arene C-99 (calix[4]arene-bis-hydroxymethylphosphonic acid) from the side of the membrane the positive potential was applied to. This current exhibited anion selectivity passing more chloride at negative potentials applied from the side of the membrane to which calix[4]arene C-99 was introduced. The kinetics and temperature-dependence determined for calix[4]arene C-99-mediated ionic transport suggest a carrier mode of facilitated diffusion.

Glutamate release from platelets: exocytosis versus glutamate transporter reversal.

Platelets express neuronal and glial glutamate transporters EAAT 1-3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-ß-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K(+)] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[(14)C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-ß-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H(+)-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K(+)], dl-THA, FCCP and bafilomycin A1. In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is rather ambiguous.