Calcium as a second messenger in neuronal growth cones

Calcium is thought to be involved in the regulation of motility and growth in the neuronal growth cone. The aim of this work was to investigate the role of cytosolic free calcium concentration ([Ca2+]i) and voltage-gated calcium influx in controlling growth cone behaviour and neurite elongation. [Ca2+]i and voltage-gated calcium influx was measured using digital imaging fluorescence microscopy (DIFM) and the whole-cell patch-clamp technique. The behaviour of growth cones of N IE-115 mouse neuroblastoma was observed and categorized into five classes. The spatial distribution of [Ca2+]i associated with these behavioural states was measured. True growth occurred only at the lowest levels. Any spontaneous elevation of [Ca2+]i above this level was associated with an inhibition of neurite elongation. Large elevations of [Ca2+]i were associated with specific morphological changes. L- and T-type voltage gated calcium currents were measured during differentiation. All cells possessing neurites had T-type currents. T-type current density was higher in cells with neurites than in cells without. There was no other detectable change in the characteristics of either current during differentiation. The spatial distribution of voltage-dependent calcium influx through L- and T-type channels was measured at the growth cone. L-type channels were found to be clustered in the growth cone membrane, whereas T-type channels were uniformly distributed. Activation of L-channel clusters caused [Ca2+]i 'hotspots' in which [Ca2+]i rose to micromolar levels during 1s depolarizations. Localized morphological changes at the site of the hotspot were observed during action potentials

Functional Properties of Dendritic Gap Junctions in Cerebellar Golgi Cells.

The strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells

Dynamic wavefront shaping with an acousto-optic lens for laser scanning microscopy

Acousto-optic deflectors (AODs) arranged in series and driven with linearly chirped frequencies can rapidly focus and tilt optical wavefronts, enabling high-speed 3D random access microscopy. Non-linearly chirped acoustic drive frequencies can also be used to shape the optical wavefront allowing a range of higher-order aberrations to be generated. However, to date, wavefront shaping with AODs has been achieved by using single laser pulses for strobed illumination to 'freeze' the moving acoustic wavefront, limiting voxel acquisition rates. Here we show that dynamic wavefront shaping can be achieved by applying non-linear drive frequencies to a pair of AODs with counter-propagating acoustic waves, which comprise a cylindrical acousto-optic lens (AOL). Using a cylindrical AOL we demonstrate high-speed continuous axial line scanning and the first experimental AOL-based correction of a cylindrical lens aberration at 30 kHz, accurate to 1/35th of a wave at 800 nm. Furthermore, we develop a model to show how spherical aberration, which is the major aberration in AOL-based remote-focusing systems, can be partially or fully corrected with AOLs consisting of four or six AODs, respectively

When do fractured media become seismically anisotropic? Some implications on quantifying fracture properties

Fractures are pervasive features within the Earth's crust and they have a significant influence on the multi-physical response of the subsurface. The presence of coherent fracture sets often leads to observable seismic anisotropy enabling seismic techniques to remotely locate and characterise fracture systems. In this study, we confirm the general scale-dependence of seismic anisotropy and provide new results specific to shear-wave splitting (SWS). We find that SWS develops under conditions when the ratio of wavelength to fracture size (λS/d) is greater than 3, where Rayleigh scattering from coherent fractures leads to an effective anisotropy such that effective medium model (EMM) theory is qualitatively valid. When 1<λS/d<3 there is a transition from Rayleigh to Mie scattering, where no effective anisotropy develops and hence the SWS measurements are unstable. When λS/d<1 we observe geometric scattering and begin to see behaviour similar to transverse isotropy. We find that seismic anisotropy is more sensitive to fracture density than fracture compliance ratio. More importantly, we observe that the transition from scattering to an effective anisotropic regime occurs over a propagation distance between 1 and 2 wavelengths depending on the fracture density and compliance ratio. The existence of a transition zone means that inversion of seismic anisotropy parameters based on EMM will be fundamentally biased. More importantly, we observe that linear slip EMM commonly used in inverting fracture properties is inconsistent with our results and leads to errors of approximately 400% in fracture spacing (equivalent to fracture density) and 60% in fracture compliance. Although EMM representations can yield reliable estimates of fracture orientation and spatial location, our results show that EMM representations will systematically fail in providing quantitatively accurate estimates of other physical fracture properties, such as fracture density and compliance. Thus more robust and accurate quantitative estimates of in situ fracture properties will require improvements to effective medium models as well as the incorporation of full-waveform inversion techniques

Geppetto: a reusable modular open platform for exploring neuroscience data and models

Geppetto is an open-source platform that provides generic middleware infrastructure for building both online and desktop tools for visualizing neuroscience models and data and managing simulations. Geppetto underpins a number of neuroscience applications, including Open Source Brain (OSB), Virtual Fly Brain (VFB), NEURON-UI and NetPyNE-UI. OSB is used by researchers to create and visualize computational neuroscience models described in NeuroML and simulate them through the browser. VFB is the reference hub for Drosophila melanogaster neural anatomy and imaging data including neuropil, segmented neurons, microscopy stacks and gene expression pattern data. Geppetto is also being used to build a new user interface for NEURON, a widely used neuronal simulation environment, and for NetPyNE, a Python package for network modelling using NEURON. Geppetto defines domain agnostic abstractions used by all these applications to represent their models and data and offers a set of modules and components to integrate, visualize and control simulations in a highly accessible way. The platform comprises a backend which can connect to external data sources, model repositories and simulators together with a highly customizable frontend.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'