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161 Publications
Showing 41-50 of 161 resultsThe need for optical sectioning in bio-imaging has amongst others led to the development of the two-photon scanning microscopy. However, this comes with some intrinsic fundamental limitations in the temporal domain as the focused spot has to be scanned mechanically in the sample plane. Hence for a large number of biological applications where imaging speed is a limiting factor, it would be significantly advantageous to generate widefield excitations with an optical sectioning comparable to the two-photon scanning microscopy. Recently by using the technique of temporal focusing it was shown that high axial resolution widefield excitation can be generated in picosecond time scales without any mechanical moving parts. However the achievable axial resolution is still well above that of a two-photon scanning microscope. Here we demonstrate a new ultrafast widefield two-photon imaging technique termed Multifocal Temporal Focusing (MUTEF) which relies on the generation of a set of diffraction limited beams produced by an Echelle grating that scan across a second tilted diffraction grating in picosecond time scale, generating a widefield excitation area with an axial resolution comparable to a two-photon scanning microscope. Using this method we have shown widefield two-photon imaging on fixed biological samples with an axial sectioning with a FWHM of 0.85 μm.
Corticosteroids (CS) act synergistically with thyroid hormone (TH) to accelerate amphibian metamorphosis. Earlier studies showed that CS increase nuclear 3,5,3’-triiodothyronine (T(3)) binding capacity in tadpole tail, and 5’ deiodinase activity in tadpole tissues, increasing the generation of T(3) from thyroxine (T(4)). In the present study we investigated CS synergy with TH by analyzing expression of key genes involved in TH and CS signaling using tadpole tail explant cultures, prometamorphic tadpoles, and frog tissue culture cells (XTC-2 and XLT-15). Treatment of tail explants with T(3) at 100 nM, but not at 10 nM caused tail regression. Corticosterone (CORT) at three doses (100, 500 and 3400 nM) had no effect or increased tail size. T(3) at 10 nM plus CORT caused tails to regress similar to 100 nM T(3). Thyroid hormone receptor beta (TRbeta) mRNA was synergistically upregulated by T(3) plus CORT in tail explants, tail and brain in vivo, and tissue culture cells. The activating 5’ deiodinase type 2 (D2) mRNA was induced by T(3) and CORT in tail explants and tail in vivo. Thyroid hormone increased expression of glucocorticoid (GR) and mineralocorticoid receptor (MR) mRNAs. Our findings support that the synergistic actions of TH and CS in metamorphosis occur at the level of expression of genes for TRbeta and D2, enhancing tissue sensitivity to TH. Concurrently, TH enhances tissue sensitivity to CS by upregulating GR and MR. Environmental stressors can modulate the timing of tadpole metamorphosis in part by CS enhancing the response of tadpole tissues to the actions of TH.
The neuroarchitecture of Acoela has been at the center of morphological debates. Some authors, using immunochemical tools, suggest that the nervous system in Acoela is organized as a commissural brain that bears little resemblance to the central, ganglionic type brain of other flatworms, and bilaterians in general. Others, who used histological staining on paraffin sections, conclude that it is a compact structure (an endonal brain; e.g., Raikova 2004; von Graff 1891; Delage Arch Zool Exp Gén 4:109-144, 1886). To address this question with modern tools, we have obtained images from serial transmission electron microscopic sections of the entire hatchling of Symsagittifera roscoffensis. In addition, we obtained data from wholemounts of hatchlings labeled with markers for serotonin and tyrosinated tubulin. Our data show that the central nervous system of a juvenile S. roscoffensis consists of an anterior compact brain, formed by a dense, bilobed mass of neuronal cell bodies surrounding a central neuropile. The neuropile flanks the median statocyst and contains several types of neurites, classified according to their types of synaptic vesicles. The neuropile issues three pairs of nerve cords that run at different dorso-ventral positions along the whole length of the body. Neuronal cell bodies flank the cords, and neuromuscular synapses are abundant. The TEM analysis also reveals different classes of peripheral sensory neurons and provides valuable information about the spatial relationships between neurites and other cell types within the brain and nerve cords. We conclude that the acoel S. roscoffensis has a central brain that is comparable in size and architecture to the brain of other (rhabditophoran) flatworms.
In the past few years, three-dimensional (3D) subtomogram alignment has become an important tool in cryo-electron tomography (CET). This technique allows one to produce higher resolution images of structures which can not be reconstructed using single-particle methods. Building on previous work, we present a new dissimilarity measure between subtomograms that works well for the noisy images that often occur in CET images. A technique that is more robust to noise provides the ability to analyze macromolecules in thicker samples such as whole cells or lower the defocus in thinner samples to push the first zero of the Contrast Transfer Function (CTF). Our method, Threshold Constrained Cross-Correlation (TCCC), uses statistics of the noise to automatically select only a small percentage of the Fourier coefficients to compute the cross-correlation, which has two main advantages: first, it reduces the influence of the noise by looking at only those peaks dominated by signal; and second, it avoids the missing wedge normalization problem since we consider the same number of coefficients for all possible pairs of subtomograms. We present results with synthetic and real data to compare our approach with other existing methods under different SNR and missing wedge conditions, and show that TCCC improves alignment results for datasets with SNR<0.1. We have made our source code freely available for the community.
Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.
Changes in behavioral state modify neural activity in many systems. In some vertebrates such modulation has been observed and interpreted in the context of attention and sensorimotor coordinate transformations. Here we report state-dependent activity modulations during walking in a visual-motor pathway of Drosophila. We used two-photon imaging to monitor intracellular calcium activity in motion-sensitive lobula plate tangential cells (LPTCs) in head-fixed Drosophila walking on an air-supported ball. Cells of the horizontal system (HS)–a subgroup of LPTCs–showed stronger calcium transients in response to visual motion when flies were walking rather than resting. The amplified responses were also correlated with walking speed. Moreover, HS neurons showed a relatively higher gain in response strength at higher temporal frequencies, and their optimum temporal frequency was shifted toward higher motion speeds. Walking-dependent modulation of HS neurons in the Drosophila visual system may constitute a mechanism to facilitate processing of higher image speeds in behavioral contexts where these speeds of visual motion are relevant for course stabilization.
The generation of coordinated body movements relies on sensory feedback from mechanosensitive proprioceptors. We have found that the proper function of NompC, a putative mechanosensitive TRP channel, is not only required for fly locomotion, but also crucial for larval crawling. Calcium imaging revealed that NompC is required for the activation of two subtypes of sensory neurons during peristaltic muscle contractions. Having isolated a full-length nompC cDNA with a protein coding sequence larger than previously predicted, we demonstrate its function by rescuing locomotion defects in nompC mutants, and further show that antibodies against the extended C terminus recognize NompC in chordotonal ciliary tips. Moreover, we show that the ankyrin repeats in NompC are required for proper localization and function of NompC in vivo and are required for association of NompC with microtubules. Taken together, our findings suggest that NompC mediates proprioception in locomotion and support its role as a mechanosensitive channel.
Continuous attractor networks are used to model the storage and representation of analog quantities, such as position of a visual stimulus. The storage of multiple continuous attractors in the same network has previously been studied in the context of self-position coding. Several uncorrelated maps of environments are stored in the synaptic connections, and a position in a given environment is represented by a localized pattern of neural activity in the corresponding map, driven by a spatially tuned input. Here we analyze networks storing a pair of correlated maps, or a morph sequence between two uncorrelated maps. We find a novel state in which the network activity is simultaneously localized in both maps. In this state, a fixed cue presented to the network does not determine uniquely the location of the bump, i.e. the response is unreliable, with neurons not always responding when their preferred input is present. When the tuned input varies smoothly in time, the neuronal responses become reliable and selective for the environment: the subset of neurons responsive to a moving input in one map changes almost completely in the other map. This form of remapping is a non-trivial transformation between the tuned input to the network and the resulting tuning curves of the neurons. The new state of the network could be related to the formation of direction selectivity in one-dimensional environments and hippocampal remapping. The applicability of the model is not confined to self-position representations; we show an instance of the network solving a simple delayed discrimination task.
To study the complex synaptic interactions underpinning dendritic information processing in single neurons, experimenters require methods to mimic presynaptic neurotransmitter release at multiple sites with no physiological damage. We show that laser scanning systems built around large-aperture acousto-optic deflectors and high numerical aperture objective lenses provide the sub-millisecond, sub-micron precision necessary to achieve physiological, exogenous synaptic stimulation. Our laser scanning systems can produce the sophisticated spatio-temporal patterns of synaptic input that are necessary to investigate single-neuron dendritic physiology.
Mitochondria are difficult targets for microscopy because of their small size and highly compartmentalized, membranous interior. Super-resolution fluorescence microscopy methods have recently been developed that exceed the historical limits of optical imaging. Here we outline considerations and techniques in preparing to image the relative location of mitochondrial proteins using photoactivated localization microscopy (PALM). PALM and similar methods have the capacity to dramatically increase our ability to image proteins within mitochondria, and to expand our knowledge of the location of macromolecules beyond the current limits of immunoEM.