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2529 Janelia Publications
Showing 1281-1290 of 2529 resultsFemtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.
A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.
Mood-altering compounds hold promise for the treatment of many psychiatric disorders, such as depression, but connecting their molecular, circuit, and behavioral effects has been challenging. Here we find that, analogous to effects in rodent learned helplessness models, ketamine pre-exposure persistently suppresses futility-induced passivity in larval zebrafish. While antidepressants are thought to primarily act on neurons, brain-wide imaging in behaving zebrafish showed that ketamine elevates intracellular calcium in astroglia for many minutes, followed by persistent calcium downregulation post-washout. Calcium elevation depends on astroglial α1-adrenergic receptors and is required for suppression of passivity. Chemo-/optogenetic perturbations of noradrenergic neurons and astroglia demonstrate that the aftereffects of glial calcium elevation are sufficient to suppress passivity by inhibiting neuronal-astroglial integration of behavioral futility. Imaging in mouse cortex reveals that ketamine elevates astroglial calcium through conserved pathways, suggesting that ketamine exerts its behavioral effects by persistently modulating evolutionarily ancient neuromodulatory systems spanning neurons and astroglia.
The brain can become transiently disconnected from the environment while maintaining vivid, internally generated experiences. This so-called 'dissociated state' can occur in pathological conditions and under the influence of psychedelics or the anesthetic ketamine (KET). The cellular and circuit mechanisms producing the dissociative state remain poorly understood. We show in mice that KET causes spontaneously active neurons to become suppressed while previously silent neurons become spontaneously activated. This switch occurs in all cortical layers and different cortical regions, is induced by both systemic and cortical application of KET and is mediated by suppression of parvalbumin and somatostatin interneuron activity and inhibition of NMDA receptors and HCN channels. Combined, our results reveal two largely non-overlapping cortical neuronal populations-one engaged in wakefulness, the other contributing to the KET-induced brain state-and may lay the foundation for understanding how the brain might become disconnected from the surrounding environment while maintaining internal subjective experiences.
Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 microns and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.
Understanding information processing in the brain requires us to monitor neural activity in vivo at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope (2PFM) empowered by all-optical laser scanning, we imaged neural activity in vivo at 1,000 frames per second and submicron spatial resolution. This ultrafast imaging method enabled monitoring of electrical activity down to 300 μm below the brain surface in head fixed awake mice.
Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.
How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that most GAF is chromatin bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for more than 2 min, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF's DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF's neighbors such as Heat Shock Factor. Evaluation of GAF kinetics together with its endogenous abundance indicates that, despite on-off dynamics, GAF constitutively and fully occupies major chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental and developmental signals.
Histone-lysine N-methyltransferase 2 (KMT2) methyltransferases play critical roles in gene regulation, cell differentiation, animal development, and human diseases. KMT2 biological roles are often attributed to their methyltransferase activities on lysine 4 of histone H3 (H3K4). However, recent data indicate that KMT2 proteins also possess non-enzymatic functions. In this review, we discuss the current understanding of KMT2 family, with a focus on their enzymatic activity-dependent and -independent functions. Six mammalian KMT2 proteins of three subgroups, KMT2A/B (MLL1/2), KMT2C/D (MLL3/4), and KMT2F/G (SETD1A/B or SET1A/B), have shared and distinct protein domains, catalytic substrates, genomic localizations, and associated complex subunits. Recent studies have revealed the central role of KMT2C/D in enhancer regulation, differentiation, and development and have highlighted KMT2C/D enzymatic activity-dependent and independent roles in mouse embryonic development and cell differentiation. Catalytic dependent and independent roles for KMT2A/B and KMT2F/G in gene regulation, differentiation, and development are less understood. Finally, we provide our perspectives and lay out future research directions that may help advance the investigation on enzymatic activity-dependent and -independent biological roles and working mechanisms of KMT2 methyltransferases.