Video_2_Computational modeling of trans-synaptic nanocolumns, a modulator of synaptic transmission.avi

Understanding synaptic transmission is of crucial importance in neuroscience. The spatial organization of receptors, vesicle release properties and neurotransmitter molecule diffusion can strongly influence features of synaptic currents. Newly discovered structures coined trans-synaptic nanocolumns were shown to align presynaptic vesicles release sites and postsynaptic receptors. However, how these structures, spanning a few tens of nanometers, shape synaptic signaling remains little understood. Given the difficulty to probe submicroscopic structures experimentally, computer modeling is a useful approach to investigate the possible functional impacts and role of nanocolumns. In our in silico model, as has been experimentally observed, a nanocolumn is characterized by a tight distribution of postsynaptic receptors aligned with the presynaptic vesicle release site and by the presence of trans-synaptic molecules which can modulate neurotransmitter molecule diffusion. In this work, we found that nanocolumns can play an important role in reinforcing synaptic current mostly when the presynaptic vesicle contains a small number of neurotransmitter molecules. Our work proposes a new methodology to investigate in silico how the existence of trans-synaptic nanocolumns, the nanometric organization of the synapse and the lateral diffusion of receptors shape the features of the synaptic current such as its amplitude and kinetics.</p

Metrological Investigation of the (6,5) Carbon Nanotube Absorption Cross Section

Using single-nanotube absorption microscopy, we measured the absorption cross section of (6,5) carbon nanotubes at their second-order optical transition. We obtained a value of 3.2 × 10^–17 cm²/C atom with a precision of 15% and an accuracy below 20%. This constitutes the first metrological investigation of the absorption cross section of chirality-identified nanotubes. Correlative absorption–luminescence microscopies performed on long nanotubes reveal a direct manifestation of exciton diffusion in the nanotube

Spatial fluorescence intensity moment analysis of the NBCe1-A oligomerization states.

<p>The percent occurrence graphs (A–C) show the recovered values of quantal brightness normalized to MEU. The experiments were carried out on fixed cells. All of the measurements summarized in each histogram were carried under identical collection conditions.</p

SpIDA measurements of NBCe1-A monomer-dimer density in expression systems (CHO-K1 cells) and native tissues.

<p>SpIDA was applied to CLSM images assuming a monomer-dimer mixture model (Equation 4). The values of monomeric, dimeric and total subunit number density were obtained for NBCe1-A in heterologous expression systems and tissues. The recovered number density of monomeric NBCe1-A in tissues is expected to be overestimated based on the presence of non-specific antibody binding. The error bars represent the standard error of the mean obtained from multiple cells. All of the measurements were carried out under identical collection conditions.</p

Sample CLSM images of CHO-K1 cells transiently transfected with NBCe1-A.

<p>(A) NBCe1-A-EGFP. (B) NBCe1-A-α-bungarotoxin mutant expressed in cells, and labeled with Alexa 488-α-bungarotoxin conjugate. In both cases, basal membranes of highly adherent cells was imaged. The cells were chemically fixed prior to imaging. The pixel size is 0.046 µm. The images were taken under the identical acquisition condition so that valid comparisons between different images could be made. The image contrast was enhanced in both panel A and B for visualization purposes.</p

Dual-color CLSM images of HEK293 cells transiently expressing NBCe1-A-EGFP immunostained with Alexa Fluor 647 conjugated antibody.

<p>Post fixation, cells were immunolabeled with the primary anti-wt-NBCe1-A antibody and the secondary Alexa Fluor 647 conjugated antibody. Panel A shows the fluorescence image taken in channel 0 (green), Panel B shows the image recorded in channel 1 (red), and Panel C is the overlap of the two. The image contrast was enhanced for better visualization of non-specific binding of the secondary Alexa Fluor 647 conjugated antibody to HEK293 cells. The pixel size is 0.0921 µm.</p

Characterization of the shot noise profiles for PMT detector.

<p>Plot of the variance as a function of the mean intensity for uniform illumination acquired with CLSM. Only the initial part of the data was taken into account for linear fitting.</p

Dependency of Cl⁻ accumulation on the site of synaptic input and KCC2 level.

<p>Trains (40 Hz) of inhibitory postsynaptic currents (IPSCs) at a synapse located at one of four positions: soma and proximal, middle, and distal dendrites (40, 100, and 240 µm from soma, respectively) in simulations without (<b>A</b>) and with (<b>B</b>) background synaptic input (<i>f</i>_inh  =  0.4 Hz, <i>f</i>_exc  =  0.1 Hz). For this set of simulations, a single dendrite was lengthened (and number of compartments increased to 60) relative to the cell geometry summarized in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002149#pcbi-1002149-g001" target="_blank">Fig. 1A</a>. Inversion of the IPSC was evident in the distal dendrites under conditions without KCC2 (<i>right panels</i>). <b>C</b>. Mean intraburst IPSC became smaller (<i>i.e.</i> less hyperpolarizing) with increasing distance from the soma and with decreasing KCC2 level. Synaptic background activity was the same as in <b>B</b>. Mean IPSC was measured at a “test” synapse activated at 40 Hz for 200 ms every second over 50 s of simulated time. Steady state value of <i>E</i>_Cl (<b>D</b>) and rate at which <i>E</i>_Cl approaches steady state (<b>E</b>) for different KCC2 levels and distances of “test” synapse from the soma. Steady state <i>E</i>_Cl reported in <b>D</b> was measured as the value to which <i>E</i>_Cl converged when GABA_AR at the test synapse were artificially held open. This convergence was fit with a single exponential to determine the time constant reported in <b>E</b>.</p

Sample CLSM images of native NBCe1-A on basolateral cell membranes of rat kidney.

<p>Post fixation, tissues were immunolabeled with the primary anti-wt-NBCe1-A antibody (I) and the secondary (II) Alexa Fluor 488 conjugated antibody. Panel A shows non-specific secondary antibody staining in the absence of the primary. Panel B shows kidney cells immunostained with both antibodies. The pixel size is 0.0921 µm. The images were taken at identical acquisition conditions so valid comparisons between different images could be made. The image contrast was enhanced for visualization and comparison purposes.</p

Redistribution of intracellular Cl⁻ through electrodiffusion.

<p><b>A.</b> Chloride influx through a single GABA_AR synapse (located at 200 µm from the soma) activated at 50 Hz produced a substantial longitudinal gradient in [Cl⁻]_i extending 60 µm on each side of the input. <b>B.</b> Chloride influx through a single GABA_AR synapse (this time located at 50 µm from the soma) produced a longitudinal gradient which is steeper toward the soma (centripetal diffusion) than away from the soma (centrifugal diffusion). For this simulation, we used dendrites with constant diameters to ensure that the difference between left and right panels is due to the sink effect of the soma and not to the conical shape of the dendrite. We also lengthened the dendrite and increased the number of compartments to 60 compared to the cell geometry summarized in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002149#pcbi-1002149-g001" target="_blank">Fig. 1</a>. <b>C.</b> Spread of Cl⁻ entering through one synapse (activated at 50 Hz) to a second “test” synapse (activated at 5 Hz) at varying inter-synapses distances was measured for normal and low (10%) KCC2 levels. Both synapses were activated simultaneously. For synapses positioned on the same primary dendrites (upper panel), the test synapse experienced a sizeable increase in [Cl⁻]_i, especially when KCC2 was reduced, but there was no appreciable spread of Cl⁻ between synapses located on different primary dendrites (lower panel).</p