A fast and robust method for automated analysis of axonal transport

Cargo movement along axons and dendrites is indispensable for the survival and maintenance of neuronal networks. Key parameters of this transport such as particle velocities and pausing times are often studied using kymograph construction, which converts the transport along a line of interest from a time-lapse movie into a position versus time image. Here we present a method for the automatic analysis of such kymographs based on the Hough transform, which is a robust and fast technique to extract lines from images. The applicability of the method was tested on simulated kymograph images and real data from axonal transport of synaptophysin and tetanus toxin as well as the velocity analysis of synaptic vesicle sharing between adjacent synapses in hippocampal neurons. Efficiency analysis revealed that the algorithm is able to detect a wide range of velocities and can be used at low signal-to-noise ratios. The present work enables the quantification of axonal transport parameters with high throughput with no a priori assumptions and minimal human intervention.     

Determination of axonal transport velocities via image cross- and autocorrelation

On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. O. Welzel and D. Boening contributed equally to this work.     

KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons

In axons, proper localization of proteins, vesicles, organelles, and other cargoes is accomplished by the highly regulated coordination of kinesins and dyneins, molecular motors that bind to cargoes and translocate them along microtubule (MT) tracks. Impairment of axonal transport is implicated in the pathogenesis of multiple neurodegenerative disorders including Alzheimer's and Huntington's diseases. To understand how MT‐based cargo motility is regulated and to delineate its role in neurodegeneration, it is critical to analyze the detailed dynamics of moving cargoes inside axons. Here, we present KymoAnalyzer, a software tool that facilitates the robust analysis of axonal transport from time‐lapse live‐imaging sequences. KymoAnalyzer is an open‐source software that automatically classifies particle trajectories and systematically calculates velocities, run lengths, pauses, and a wealth of other parameters that are characteristic of motor‐based transport. We anticipate that laboratories will easily use this package to unveil previously uncovered intracellular transport details of individually‐moving cargoes inside neurons.      

Ferlins Show Tissue‐Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans‐Golgi/Recycling Ferlins

Ferlins are a family of transmembrane‐anchored vesicle fusion proteins uniquely characterized by 5–7 tandem cytoplasmic C2 domains, Ca²⁺‐regulated phospholipid‐binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb‐girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca²⁺‐dependent, vesicle‐mediated membrane repair and otoferlin mutations cause non‐syndromic deafness due to defective Ca²⁺‐triggered auditory neurotransmission. In this study, we describe the tissue‐specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D‐structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7‐positive late endosomes, supporting potential roles in the late‐endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans‐Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans‐Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type‐I and type‐II ferlins segregate as PM/late‐endosomal or trans‐Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.      

CDK5‐dependent activation of dynein in the axon initial segment regulates polarized cargo transport in neurons

The unique polarization of neurons depends on selective sorting of axonal and somatodendritic cargos to their correct compartments. Axodendritic sorting and filtering occurs within the axon initial segment (AIS). However, the underlying molecular mechanisms responsible for this filter are not well understood. Here, we show that local activation of the neuronal‐specific kinase cyclin‐dependent kinase 5 (CDK5) is required to maintain AIS integrity, as depletion or inhibition of CDK5 induces disordered microtubule polarity and loss of AIS cytoskeletal structure. Furthermore, CDK5‐dependent phosphorylation of the dynein regulator Ndel1 is required for proper re‐routing of mislocalized somatodendritic cargo out of the AIS; inhibition of this pathway induces profound mis‐sorting defects. While inhibition of the CDK5‐Ndel1‐Lis1‐dynein pathway alters both axonal microtubule polarity and axodendritic sorting, we found that these defects occur on distinct timescales; brief inhibition of dynein disrupts axonal cargo sorting before loss of microtubule polarity becomes evident. Together, these studies identify CDK5 as a master upstream regulator of trafficking in vertebrate neurons, required for both AIS microtubule organization and polarized dynein‐dependent sorting of axodendritic cargos, and support an ongoing and essential role for dynein at the AIS.      

Origin of improved depth penetration in dual‐axis optical coherence tomography: a Monte Carlo study

Recent studies have demonstrated that extended imaging depth can be achieved using dual‐axis optical coherence tomography (DA‐OCT). By illuminating and collecting at an oblique angle, multiple forward scattered photons from large probing depths are preferentially detected. However, the mechanism behind the enhancement of imaging depth needs further illumination. Here, the signal of a DA‐OCT system is studied using a Monte Carlo simulation. We modeled light transport in tissue and recorded the spatial and angular distribution of photons exiting the tissue surface. Results indicate that the spatial separation and offset angle created by the non‐telecentric scanning configuration promote the collection of more deeply propagating photons than conventional on‐axis OCT.      

miR‐181a/b control the assembly of visual circuitry by regulating retinal axon specification and growth

Connectivity and function of neuronal circuitry require the correct specification and growth of axons and dendrites. Here, we identify the microRNAs miR‐181a and miR‐181b as key regulators of retinal axon specification and growth. Loss of miR‐181a/b in medaka fish (Oryzias latipes) failed to consolidate amacrine cell processes into axons and delayed the growth of retinal ganglion cell (RGC) axons. These alterations were accompanied by defects in visual connectivity and function. We demonstrated that miR‐181a/b exert these actions through negative modulation of MAPK/ERK signaling that in turn leads to RhoA reduction and proper neuritogenesis in both amacrine cells and RGCs via local cytoskeletal rearrangement. Our results identify a new pathway for axon specification and growth unraveling a crucial role of miR‐181a/b in the proper establishment of visual system connectivity and function. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1252–1267, 2015     

Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co‐regulate axonal outgrowth of mouse retinal ganglion cells

Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc‐dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad‐spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP‐deficient mice, disclosed that both MMP‐2 and MT1‐MMP, but not MMP‐9, are involved in this process. Furthermore, administration of a novel antibody to MT1‐MMP that selectively blocks pro‐MMP‐2 activation revealed a functional co‐involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP‐2 and MT1‐MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP‐2 and β1‐integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP‐2 and MT1‐MMP as promising axonal outgrowth‐promoting molecules.

     

Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

Summary The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3-diaminobenzidine (DAB) chromogen.The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 m) and semithin (1 m) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.Supported by NIH Grant NS19266     

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein‐regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth‐associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

     

Role of an adaptor protein Lin‐7B in brain development: possible involvement in autism spectrum disorders

Using comparative genomic hybridization analysis for an autism spectrum disorder (ASD) patient, a 73‐Kb duplication at 19q13.33 (nt. 49 562 755–49 635 956) including LIN7B and 5 other genes was detected. We then identified a novel frameshift mutation in LIN7B in another ASD patient. Since LIN7B encodes a scaffold protein essential for neuronal function, we analyzed the role of Lin‐7B in the development of cerebral cortex. Acute knockdown of Lin‐7B with in utero electroporation caused a delay in neuronal migration during corticogenesis. When Lin‐7B was knocked down in cortical neurons in one hemisphere, their axons failed to extend efficiently into the contralateral hemisphere after leaving the corpus callosum. Meanwhile, enhanced expression of Lin‐7B had no effects on both cortical neuron migration and axon growth. Notably, silencing of Lin‐7B did not affect the proliferation of neuronal progenitors and stem cells. Taken together, Lin‐7B was found to play a pivotal role in corticogenesis through the regulation of excitatory neuron migration and interhemispheric axon growth, while further analyses are required to directly link functional defects of Lin‐7B to ASD pathophysiology.

     

Sensitivity and accuracy of hybrid fluorescence‐mediated tomography in deep tissue regions

Fluorescence‐mediated tomography (FMT) enables noninvasive assessment of the three‐dimensional distribution of near‐infrared fluorescence in mice. The combination with micro‐computed tomography (µCT) provides anatomical data, enabling improved fluorescence reconstruction and image analysis. The aim of our study was to assess sensitivity and accuracy of µCT‐FMT under realistic in vivo conditions in deeply‐seated regions. Accordingly, we acquired fluorescence reflectance images (FRI) and µCT‐FMT scans of mice which were prepared with rectal insertions with different amounts of fluorescent dye. Default and high‐sensitivity scans were acquired and background signal was analyzed for three FMT channels (670 nm, 745 nm, and 790 nm). Analysis was performed for the original and an improved FMT reconstruction using the µCT data. While FRI and the original FMT reconstruction could detect 100 pmol, the improved FMT reconstruction could detect 10 pmol and significantly improved signal localization. By using a finer sampling grid and increasing the exposure time, the sensitivity could be further improved to detect 0.5 pmol. Background signal was highest in the 670 nm channel and most prominent in the gastro‐intestinal tract and in organs with high relative amounts of blood. In conclusion, we show that µCT‐FMT allows sensitive and accurate assessment of fluorescence in deep tissue regions.

     

Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488‐conjugated cholesterol molecule (Bdp‐Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent‐molecule imaging. Surprisingly, in the intact PM, Bdp‐Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm²/second, which was ～10 times greater than that of non‐raft phospholipid molecules (0.33 µm²/second), despite Bdp‐Chol's probable association with raft domains. Furthermore, Bdp‐Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp‐Chol and Cy3‐conjugated dioleoylphosphatidylethanolamine (Cy3‐DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm²/second, respectively), indicating that the actin‐based membrane skeleton reduces the D of Bdp‐Chol only by a factor of ～2 from that in the blebbed PM, whereas it reduces the D of Cy3‐DOPE by a factor of ～20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin‐based membrane‐skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp‐Chol passing through the compartment boundaries, once it enters the boundary, is ～10× greater than that of Cy3‐DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane‐skeleton‐induced compartments and are contained within them.      

Deep learning‐based color holographic microscopy

We report a framework based on a generative adversarial network that performs high‐fidelity color image reconstruction using a single hologram of a sample that is illuminated simultaneously by light at three different wavelengths. The trained network learns to eliminate missing‐phase‐related artifacts, and generates an accurate color transformation for the reconstructed image. Our framework is experimentally demonstrated using lung and prostate tissue sections that are labeled with different histological stains. This framework is envisaged to be applicable to point‐of‐care histopathology and presents a significant improvement in the throughput of coherent microscopy systems given that only a single hologram of the specimen is required for accurate color imaging.     

Identification of precursor microRNAs within distal axons of sensory neuron

A set of specific precursor microRNAs (pre‐miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre‐miRNAs are also transported into distal axons to autonomously regulate intra‐axonal protein synthesis. Here, we show that a subset of pre‐miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre‐miRNAs (let 7c‐a and pre‐miRs‐16, 23a, 25, 125b‐1, 433, and 541) showed elevated axonal levels, while others (pre‐miRs‐138‐2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre‐miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.

     

Front Cover: A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution (J. Biophotonics 2/2018)

A new type of high‐throughput imaging flow cytometer (>20 000 cells s^‐1) based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. FACED imaging flow cytometers enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. It critically empowers largescale and deep characterization of single cells and their heterogeneity with high statistical power— an ability to become increasingly critical in single‐cell analysis adopted in a wide range of biomedical and life‐science applications. Further details can be found in the article by Wenwei Yan et al. ( e201700178 )

     

Oxygenation measurement by multi‐wavelength oxygen‐dependent phosphorescence and delayed fluorescence: catchment depth and application in intact heart

Oxygen delivery and metabolism represent key factors for organ function in health and disease. We describe the optical key characteristics of a technique to comprehensively measure oxygen tension (PO₂) in myocardium, using oxygen‐dependent quenching of phosphorescence and delayed fluorescence of porphyrins, by means of Monte Carlo simulations and ex vivo experiments. Oxyphor G2 (microvascular PO₂) was excited at 442 nm and 632 nm and protoporphyrin IX (mitochondrial PO₂) at 510 nm. This resulted in catchment depths of 161 (86) µm, 350 (307) µm and 262 (255) µm respectively, as estimated by Monte Carlo simulations and ex vivo experiments (brackets). The feasibility to detect changes in oxygenation within separate anatomical compartments is demonstrated in rat heart in vivo.

Schematic of ex vivo measurements.     

Effects of the murine skull in optoacoustic brain microscopy

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small‐animal neuroimaging methods, a variety of acoustic and light‐related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high‐resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically‐ and optically‐determined resolution scenarios. It is shown that strong low‐pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

(a) Experimental setup for hybrid acoustic and optical resolution optoacoustic microscopy. (b) Transcranial scan of an adult mouse brain using the optical resolution mode. Scale bar is 375 µm.