Inhibition of Protein Synthesis Attenuates Formation of Traumatic Memory and Normalizes Fear-Induced c-Fos Expression in a Mouse Model of Posttraumatic Stress Disorder.

Posttraumatic stress disorder (PTSD) is a debilitating psychosomatic condition characterized by impairment of brain fear circuits and persistence of exceptionally strong associative memories resistant to extinction. In this study, we investigated the neural and behavioral consequences of inhibiting protein synthesis, a process known to suppress the formation of conventional aversive memories, in an established PTSD animal model based on contextual fear conditioning in mice. Control animals were subjected to the conventional fear conditioning task. Utilizing c-Fos neural activity mapping, we found that the retrieval of PTSD and normal aversive memories produced activation of an overlapping set of brain structures. However, several specific areas, such as the infralimbic cortex and the paraventricular thalamic nucleus, showed an increase in the PTSD group compared to the normal aversive memory group. Administration of protein synthesis inhibitor before PTSD induction disrupted the formation of traumatic memories, resulting in behavior that matched the behavior of mice with usual aversive memory. Concomitant with this behavioral shift was a normalization of brain c-Fos activation pattern matching the one observed in usual fear memory. Our findings demonstrate that inhibiting protein synthesis during traumatic experiences significantly impairs the development of PTSD in a mouse model. These data provide insights into the neural underpinnings of protein synthesis-dependent traumatic memory formation and open prospects for the development of new therapeutic strategies for PTSD prevention.

Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene (Plaur) and Its Molecular Target Mef2d.

Urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored receptor of urokinase (uPA), which is involved in brain development, nerve regeneration, wound healing and tissue remodeling. We have recently shown that Plaur, which encodes uPAR, is an early response gene in murine brain. Assumingly, diverse functions of Plaur might be attributed to hypothetical, unidentified microRNAs encoded within introns of the Plaur gene. Using a bioinformatic approach we identified novel small RNAs within the Plaur gene and named them Plaur-miR1-3p and Plaur-miR1-5p. We confirmed Plaur-dependent expression of Plaur-miR1-3p and Plaur-miR1-5p in the mouse brain and mouse neuroblastoma Neuro2a cells. Utilizing an in silico MR-microT algorithm in DianaTools we selected two target genes - Mef2d and Emx2 with the highest binding scores to small RNAs selected from identified Plaur-Pre-miR1. Furthermore, sequencing of mouse brain samples for Plaur-miR1-5p target genes revealed two more genes-Nrip3 and Snrnp200. The expression of Emx2, Mef2d, and Snrnp200 in the mouse brain and Mef2d and Snrnp200 in Neuro2a cells correlated with expression of Plaur and small RNAs-Plaur-miR1-3p and Plaur-miR1-5p. Finally, we demonstrated elevated MEF2D protein expression in the mouse brain after Plaur induction and displayed activating effects of Plaur-miR1-5p on Mef2d expression in Neuro2a cells using Luciferase reporter assay. In conclusion, we have identified Plaur-miR1-3p and Plaur-miR1-5p as novel small RNAs encoded in the Plaur gene. This finding expands the current understanding of Plaur function in brain development and functioning.

Implantable graded-index fibers for neural-dynamics-resolving brain imaging in awake mice on an air-lifted platform.

We demonstrate a versatile framework for cellular brain imaging in awake mice based on suitably tailored segments of graded-index (GRIN) fiber. Closed-form solutions to ray-path equations for graded-index waveguides are shown to offer important insights into image-transmission properties of GRIN fibers, suggesting useful recipes for optimized GRIN-fiber-based deep-brain imaging. We show that the lengths of GRIN imaging components intended for deep-brain studies in freely moving rodents need to be chosen as a tradeoff among the spatial resolution, the targeted imaging depth and the degree of fiber-probe invasiveness. In the experimental setting that we present in this paper, the head of an awake mouse with a GRIN-fiber implant is fixed under a microscope objective, but the mouse is free to move around an in-house-built flat-floored air-lifted platform, exploring a predesigned environment, configured as an arena for one of standard cognitive tests. We show that cellular-resolution deep-brain imaging can be integrated in this setting with robust cell-specific optical neural recording to enable in vivo studies with minimal physical restraints on animal models. The enhancement of the information capacity of the fluorescence signal, achieved via a suitable filtering of the GRIN-fiber readout, is shown to open routes toward practical imaging modalities whereby the deep-brain neuronal dynamics and axonal connections underpinning the integrative functions of essential brain structures can be studied in awake rodent models.

The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2-3- and 5-7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.

Early Induction of Neurotrophin Receptor and miRNA Genes in Mouse Brain after Pentilenetetrazole-Induced Neuronal Activity.

Neurotrophin receptors regulate neuronal survival and network formation, as well as synaptic plasticity in the brain via interaction with their ligands. Here, we examined early changes in the expression of neurotrophin receptor genes Ntk1 (TrkA), Ntrk2 (TrkB), Ntrk3 (TrkC), Ngfr (p75NTR) and miRNAs that target theses gens in the mouse brain after induction of seizure activity by pentylenetetrazol. We found that expression of Ntrk3 and Ngfr was upregulated in the cortex and the hippocampus 1-3 hours after the seizures, while Ntrk2 expression increased after 3-6 hours in the anterior cortex and after 1 and 6 hours in the hippocampus. At the same time, the ratio of Bcl-2/Bax signaling proteins increased in the anterior and posterior cortex, but not in the hippocampus, suggesting the activation of anti-apoptotic signaling. Expression of miRNA-9 and miRNA-29a, which were predicted to target Ntrk3, was upregulated in the hippocampus 3 hours after pentylenetetrazol injection. Therefore, early cellular response to seizures in the brain includes induction of the Ntrk2, Ntrk3, Ngfr, miRNA-9, and miRNA-29a expression, as well as activation of Bcl-2 and Bax signaling pathways, which may characterize them as important mediators of neuronal adaptation and survival upon induction of the generalized brain activity.

LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift.

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory.

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.

FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus.

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4-6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3-3.5, respectively.

Multisite cell- and neural-dynamics-resolving deep brain imaging in freely moving mice with implanted reconnectable fiber bundles.

We demonstrate a reconnectable implantable ultraslim fiber-optic microendoscope that integrates a branching fiber bundle (BFB) with gradient-index fiber lenses, enabling a simultaneous fluorescence imaging of individual cells in distinctly separate brain regions, including brain structures as distant as the neocortex and hippocampus. We show that fluorescence images of individual calcium-indicator-expressing neurons in the brain of freely moving transgenic mice can be recorded, via the implanted BFB probe, in parallel with time- and cell-resolved traces of calcium signaling, thus enabling correlated circuit-dynamics studies at -multiple sites within the brain of freely moving animals.

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein.

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.

Urokinase receptor and tissue plasminogen activator as immediate-early genes in pentylenetetrazole-induced seizures in the mouse brain.

Epileptogenesis progressively leads to the rearrangement of normal neuronal networks into more excitable ones and can be viewed as a form of neuroplasticity, the molecular mechanisms of which still remain obscure. Here, we studied pentylenetetrazole seizure-induced regulation of genes for plasminogen activator system in the mouse brain. We found that expression of tissue plasminogen activator (tPA) and urokinase receptor (uPAR) mRNA was strongly increased in the mouse cerebral cortex, hippocampus, striatum and amygdala as early as 3 hr after pentylenetetrazole seizures. Such early activity-induced expression of uPAR in the central nervous system has not been demonstrated before. uPAR mRNA accumulation was followed by elevation of uPAR protein, indicating a complete transcription-translation process. Both tPA gene induction and uPAR gene induction were independent of the protein synthesis, suggesting that they are regulated by neural activity as immediate-early genes. In contrast to tPA and uPAR genes, the expression of which returned to the basal level 6 hr following seizures, urokinase and plasminogen activator inhibitor-1 gene expression showed a delayed activation only at 3 days after seizures. In conclusion, our results suggest an important sensitivity of the brain plasminogen activator system to seizure activity which raises the question of its role in activity-dependent neural tissue remodeling in pathological and normal conditions.

NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics.

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.

Ionization penalty in nonlinear Raman neuroimaging.

Light-assisted ionization accompanying coherent anti-Stokes Raman scattering (CARS) of ultrashort laser pulses in brain tissue is shown to manifest itself in a detectable blueshift of the anti-Stokes signal. This blueshift can serve as an indicator of ionization processes in CARS-based neuroimaging.

Fiber-optic probes for in vivo depth-resolved neuron-activity mapping.

Specialty fiber probes are used for in vivo depth-resolved mapping of neuron activity through the optical detection of fluorescent-protein reporters expressed inside the living brain of anesthetized transgenic mice. Supercontinuum radiation produced by highly nonlinear photonic-crystal fibers is employed to demonstrate a simultaneous multicolor interrogation of several biomarkers in a model aqueous solution system, thus suggesting the way toward a multiplex mapping of various types of neuron dynamics inside the living brain.

Tailoring the soliton output of a photonic crystal fiber for enhanced two-photon excited luminescence response from fluorescent protein biomarkers and neuron activity reporters.

Dual-cladding photonic crystal fibers (PCFs) with two zero-dispersion points are used to enhance the two-photon excited luminescence (TPL) response from fluorescent protein biomarkers and neuron activity reporters in dye-cell experiments and in in vivo work on transgenic mice and tadpoles. The soliton transmission of ultrashort pulses through a PCF suppresses dispersion-induced temporal pulse spreading, maintaining a high level of field intensity needed for efficient TPL excitation. The soliton self-frequency shift, stabilized against laser power fluctuations by a specific PCF dispersion design, is employed to accurately match the wavelength of the soliton PCF output with the two-photon absorption spectrum of dye or fluorescent protein biomarker molecules, enhancing their TPL response and allowing the laser damage of biotissues to be avoided.