Extraction of sub-microscopic Ca fluxes from blurred and noisy fluorescent indicator images with a detailed model fitting approach

The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This ‘forward method’ allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes

Modelling active zone calcium dynamics at cerebellar mossy fibre boutons

The rate at which signals can be transmitted between single neurons limits the speed of information processing. Cerebellar mossy fibre boutons are able to maintain synchronous neurotransmitter release at very high action potential frequencies, up to ∼ 1 kHz . The neurotransmitter release occurs at the presynaptic active zone and is controlled by highly localised calcium signals. In order to allow reliable, fast synaptic transmission, calcium ions must be cleared from the active zone. However, the exact mechanisms of calcium clearance remain elusive. Despite the recent advances in imaging technology, it is not yet possible to measure localised calcium signals on the nanometre scale. Nevertheless, it is possible to address the impact of localised calcium signals on neurotransmitter release with use of computational modelling. In this study, I established an experimentally constrained model of an active zone of the cerebellar mossy fibre bouton. My simulations revealed that endogenous fixed buffers that have low calcium binding capacity ( ∼ 15 ) and low affinity for binding calcium in combination with mobile buffers with high affinity for binding calcium enable rapid clearance of calcium from the active zone during high-frequency firing. Moreover, during high-frequency firing, slow endogenous mobile buffers prevent build-up of the intracellular calcium concentration. The results presented in this work suggest that reduced calcium buffering speeds active zone calcium signalling, thus allowing high rates of synaptic transmission