Differences in transmission properties and susceptibility to long-term depression reveal functional specialization of ascending axon and parallel fiber synapses to Purkinje cells

An understanding of the patterns of mossy fiber transmission to Purkinje cells, via granule cell axons, is fundamental to models of cerebellar cortical signaling and processing. Early theories assumed that mossy fiber input is widely disseminated across the cerebellar cortex along beams of parallel fibers, which spread for several millimeters across the cerebellar cortex. Direct evidence for this has, however, proved controversial, leading to the development of an alternative hypothesis that mossy fiber inputs to the cerebral cortex are in fact vertically organized such that the ascending segment of the granule axon carries a greater synaptic weight than the parallel fiber segment. Here, we report that ascending axon synapses are selectively resistant to cerebellar long-term depression and that they release transmitter with higher mean release probabilities and mean quantal amplitudes than parallel fiber synapses. This novel specialization of synapses formed by different segments of the same axon not only explains the reported patterns of granule cell→ Purkinje cell transmission across the cerebellar cortex but also reveals an additional level of functionality and complexity of cerebellar processing. Consequently, ascending axon synapses represent a new element of cortical signal processing that should be distinguished from parallel fiber synapses in future experimental and theoretical studies of cerebellar function

Simple windows based software for the control of laser scanning confocal microscopes.

Rapid advances in computer processing power and the appearance of low cost, high speed multifunction data acquisition hardware makes the control of confocal laser scanning microscopes (CLSMs) with standard laboratory hardware a potentially straightforward task. This paper describes software designed to control a Biorad MRC 600 scan head under Windows 2000 or XP. Using a single high speed, multifunction data acquisition board running under the Igor Pro software environment, waveforms required to drive the scan head galvanometers can be generated and up to two channels of images (768 × 512 pixels at 8 or 12 bit levels) captured live or at set intervals. Image averaging, zooming, panning and cropping are supported as is live region of interest measurements over time. The software can trigger or be triggered by external devices via TTL signals and, with the addition of a commercial focus controller, Z scans can also be made. Control of the original neutral density and emission filters of multiple laser-based systems is also supported via serial control. The software should be easily adaptable to control custom designed scanning systems or other older makes of CLSM and it can be integrated with additional acquisition boards for simultaneous electrophysiological recording

Programmable Illumination and High-Speed, Multi- Wavelength, Confocal Microscopy Using a Digital Micromirror

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor

Differential susceptibility to synaptic plasticity reveals a functional specialization of ascending axon and parallel fiber synapses to cerebellar Purkinje cells

Granule cell axons, via their parallel fibers, form synapses with Purkinje cells across large areas of the cerebellar cortex. Evidence for uniform transmission along parallel fibers to Purkinje cells is controversial, however, leading to speculation that the ascending axonal segment plays a dominant role in cerebellar processing. We have compared the relative susceptibilities of ascending axon and parallel fiber synaptic inputs to several forms of synaptic plasticity. We demonstrate that ascending axon synapses have a limited capability to undergo forms of long-term depression and potentiation compared with parallel fiber synapses. These results demonstrate that these two segments of the same axon play fundamentally different roles in cerebellar signaling, and, as such, the synapses formed between granule cells and Purkinje cells should not be treated as a homogenous population

Characterisation of axial resolution.

<p>A. Maximum projection of wide-field images of a 4 µm bead taken over an axial distance of 120 µm at 0.5 µm intervals. Below is an axial projection from a frontal plane. B. Images taken of the same bead using a 4×6 pinhole configuration and processed in the same way. Axial projections were made for a range of different pinhole configurations and line profiles drawn through the centre of the bead. C. Axial line profiles comparing 4 different pinhole configurations with images taken in wide-field are shown. Data were fitted with a Gaussian curve to estimate the axial height of the bead. Figures D–F illustrate the relationship between axial resolution, pinhole size and pinhole separation.</p

Photoactivation of mEOS2.

<p>A layer of bacteria expressing mEO2 was photo-activated with 405 nm light. The image on the left shows the resulting loss of green fluorescence where the photoactivation took place. The image on the right shows the appearance of red fluorescence.</p

Examples of wide-field and confocal images of biological specimens.

<p>A. Wide-field (left) and confocal images (right) of cucurbita pollen grains. Excitation wavelengths were 470±nm and 560±20 nm and emission wavelengths of 500–540 nm and 600–670 nm were collected. Images represent maximum projections of stacks of images taken in the axial plane. B. Wide-field and confocal images of a snail neuron filled with Alexa Fluor 568. A cyan look up table was used for clarity. C. A comparison of cucurbita maximum projections captured with the DMD confocal (top) and Leica SP2 CLSM (bottom) with 60 and 63× objectives with N.A of 1 and 0.9 respectively D. Axial profiles of 4 µm beads measured with the DMD confocal with a 4×8 pinhole configuration and Leica SP2 CLSM with an optimal pinhole setting of 1 airy unit.</p