Super resolution imaging of genetically labeled synapses in drosophila brain tissue

Understanding synaptic connectivity and plasticity within brain circuits and their relationship to learning and behavior is a fundamental quest in neuroscience. Visualizing the fine details of synapses using optical microscopy remains however a major technical challenge. Super resolution microscopy opens the possibility to reveal molecular features of synapses beyond the diffraction limit. With direct stochastic optical reconstruction microscopy, dSTORM, we image synaptic proteins in the brain tissue of the fruit fly, Drosophila melanogaster. Super resolution imaging of brain tissue harbors difficulties due to light scattering and the density of signals. In order to reduce out of focus signal, we take advantage of the genetic tools available in the Drosophila and have fluorescently tagged synaptic proteins expressed in only a small number of neurons. These neurons form synapses within the calyx of the mushroom body, a distinct brain region involved in associative memory formation. Our results show that super resolution microscopy, in combination with genetically labeled synaptic proteins, is a powerful tool to investigate synapses in a quantitative fashion providing an entry point for studies on synaptic plasticity during learning and memory formation

List of different simulation conditions applied.

<p>All the simulations had a tonic firing of 5.6 Hz and release probability of 0.5 during the first 4 sec. For the following phasic activity, different firing rates were applied. For every condition A–D, 10, 25 or 50% of the release sites showed phasic or depressed activity.</p

Tonic release of dopamine reaches a steady state level of 26 nM.

<p><b>A.</b> Simulation volume with edge length of 64 µm. Dots indicate the location of dopamine release sites in the volume. The density of release sites is given by our previous anatomical measurements. The random distribution has been derived from the nearest neighbour analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071615#pone.0071615-Martin1" target="_blank">[15] </a><b>B.</b> A section through the simulation volume shows local ‘hot-spots’ of dopamine concentration emerging around release sites during tonic firing. The level of dopamine concentration is indicated by different colors. The release site lying within the same plane of section is indicated with a circle. Scale bar = 10 µm <b>C.</b> The plot shows the dopamine concentration along a sample line in the simulation volume at steady state. <b>D.</b> The dopamine concentration of every compartment (1 µm³) is plotted and shows the distribution and range of dopamine concentration across the entire volume at steady state. All sites receive enough dopamine to bind on D1 receptors in their high affinity state. The box is limited by the 25^th and 75^th percentiles with the median shown as red line and outliers are plotted as red crosses.</p

Summary of the simulation results obtained from the different protocols.

<p>The absolute values of the dopamine concentration at steady state (tonic firing) and after phasic firing (t = 150 ms), the absolute difference in dopamine concentration between tonic and phasic firing and the relative difference in dopamine concentration after phasic release are listed as mean with standard deviation (std). The range is presented with the minima and maxima values.</p

Simulation performed with blocked dopamine re-uptake.

<p>The mean dopamine concentration at a distance of 1 µm (red), 2 µm (yellow) and 5 µm (green) from a release site is plotted against time. The dashed lines indicate the standard deviation. The local dopamine concentration reaches steady state due to tonic release and linear re-uptake of dopamine. At time t = 1.0 sec all the re-uptake is blocked while the release sites keep their tonic release activity. The dopamine concentration increases on average 39 nM per second and would reach µM level after 20–25 seconds.</p

Local change in dopamine concentration from steady state after phasic activity plotted along a sample line through the simulation volume.

<p><b>A.</b> Local dopamine concentration is plotted at steady state (black) and after 150 ms of increased (orange: 15 Hz, red: 26 Hz) or depressed (blue, 0 Hz) firing rate along a random line of the simulation volume. <b>B.</b> The local difference in absolute dopamine concentration between steady state and that resulting from increased or decreased activity is plotted for the different protocols. <b>C.</b> The local difference in the dopamine concentration is plotted as percentage to the baseline level.</p

List of parameters applied in the simulation of dopamine diffusion and their references.

<p>List of parameters applied in the simulation of dopamine diffusion and their references.</p

Comparison of local dopamine concentrations at steady state between the two conditions with normal and highly reduced number of release sites.

<p>Simulation shows the steady state difference of local dopamine concentration for a normal density of dopamine release sites and with highly reduced number of release sites. A) Histogram shows the distribution of local dopamine concentration in the two simulation conditions. Most sites within the volume in the MPTP condition show a dopamine concentration lower than 10 nM while in the normal condition most sites are above 10 nM. B) A random sample line within the simulation volume shows the local dopamine concentration and its local variation. The black sample line is taken in the simulation volume with normal number of release sites and the grey line is sampled from the simulation volume with highly reduced numbers of release sites.</p

Change in dopamine concentration between steady state and phasic activity within the entire simulation volume.

<p>Simulations were performed with 50% of the release sites firing at 15 Hz (A), 26 Hz (B) and 0 Hz (C) during 150 ms. <b>A–C. 1</b> The distribution of dopamine concentration during steady state and after 150 ms of phasic activity is plotted (resolution: µm³). <b>A–C. 2</b> Histogram of the dopamine concentration in the volume of the lower nM range, containing most values. The distribution of local dopamine concentration is plotted during tonic activity at steady state (black) and after phasic activity (grey). <b>A–C. 3</b> Change in absolute dopamine concentration in the simulation volume for every µm³ after phasic activity. <b>A–C. 4</b>. The relative increase of the dopamine concentration after phasic activity is plotted for the different protocols as percentage of steady state.</p