jULIEs: nanostructured polytrodes for low traumatic extracellular recordings and stimulation in the mammalian brain

Objective.Extracellular microelectrode techniques are the most widely used approach to interrogate neuronal populations. However, regardless of the manufacturing method used, damage to the vasculature and circuit function during probe insertion remains a concern. This issue can be mitigated by minimising the footprint of the probe used. Reducing the size of probes typically requires either a reduction in the number of channels present in the probe, or a reduction in the individual channel area. Both lead to less effective coupling between the probe and extracellular signals of interest.Approach.Here, we show that continuously drawn SiO2-insulated ultra-microelectrode fibres offer an attractive substrate to address these challenges. Individual fibres can be fabricated to >10 m continuous stretches and a selection of diameters below 30µm with low resistance (<100 Ω mm-1) continuously conductive metal core of <10µm and atomically flat smooth shank surfaces. To optimize the properties of the miniaturised electrode-tissue interface, we electrodeposit rough Au structures followed by ∼20 nm IrOx film resulting in the reduction of the interfacial impedance to <500 kΩ at 1 kHz.Main results. We demonstrate that these ultra-low impedance electrodes can record and stimulate both single and multi-unit activity with minimal tissue disturbance and exceptional signal-to-noise ratio in both superficial (∼40µm) and deep (∼6 mm) structures of the mouse brain. Further, we show that sensor modifications are stable and probe manufacturing is reproducible.Significance.Minimally perturbing bidirectional neural interfacing can reveal circuit function in the mammalian brainin vivo

Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.

Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.

Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis

Optical Microwell Assay of Membrane Transport Kinetics

In optical single transporter recording, membranes are firmly attached to flat solid substrates containing small wells or test compartments (TC). Transport of fluorescent molecules through TC-spanning membrane patches is induced by solution change and recorded by confocal microscopy. Previously, track-etched membrane filters were used to create solid substrates containing populations of randomly distributed TCs. In this study the possibilities offered by orderly TC arrays as created by laser microdrilling were explored. A theoretical framework was developed taking the convolution of membrane transport, solution change, and diffusion into account. The optical properties of orderly TC arrays were studied and the kinetics of solution change measured. Export and import through the nuclear pore complex (NPC) was analyzed in isolated envelopes of Xenopus oocyte nuclei. In accordance with previous reports nuclear transport receptor NTF2, which binds directly to NPC proteins, was found to be translocated much faster than “inert” molecules of similar size. Unexpectedly, NXT1, a homolog of NTF2 reportedly unable to bind to NPC proteins directly, was translocated as fast as NTF2. Thus, microstructured TC arrays were shown to provide optical single transporter recording with a new basis

Comparison of mobilities of WPB proteins in NH₄Cl-rounded and post-fusion WPBs.

a<p><i>n</i>_mob and <i>n</i>_im are numbers of WPB experiments where mobile or immobile behaviour was observed, correspondingly.</p>b<p><i>D</i> and MF (mobile fraction) values as calculated for all experiments, immobile values regarded as 0s.</p>c<p>the values in square brackets single out mobile cases only.</p><p>Comparison of mobilities of WPB proteins in NH₄Cl-rounded and post-fusion WPBs.</p

Modelling post-bleaching diffusion inside a sphere: Monte-Carlo simulations and direct calculations.

<p><i>A</i>, Graphical representation of the bleaching experiment, showing an impermeable sphere of unit radius, in which the horizontal polar axis distance <i>µ</i> = cos(<i>θ</i>) and the normalised radius <i>r</i> are spherical coordinates describing fluorophore concentration distribution. A bleached region covering the horizontal axis interval <i>α</i>≤<i>µ</i>≤1 is shown in white, the rest of the sphere contains fluorophore (grey). <i>B–C</i> Monte-Carlo FRAP recovery simulations. <i>B,</i> Concentration profiles for simulated redistribution of 20000 particles, initially positioned at time <i>τ</i> = 0 s in the left hemisphere (black trace). Recovery times are coded by the trace colours. Simulation parameters were: sphere radius <i>R</i>₀ = 1 µm, prescribed diffusion coefficient <i>D</i> = 3 µm²/s, bleaching parameter <i>α</i> = 0, simulation step 5·10⁻⁴s. Profiles were constructed from images representing the 2D projections of the sphere, each simulated particle was represented by a pixel of value 1. The plot of normalised moment kinetics is shown in <i>C</i>. Red line- monoexponential fit, the fitted time constant <i>τ</i> = 83 ms corresponds to the <i>D</i> value of 2.79 µm²/s (Eq.8). <i>E–F,</i> Analysis of the same problem from images representing projections of concentration distributions calculated using Eq.10. <i>E</i>, Concentration profiles for simulated distributions, time-dependent colour coding as in <i>B</i>. Oscillating patterns (Gibb’s phenomena) at short times (black trace) are due to approximation of discontinuity by finite Legendre sums. <i>F,</i> The normalised moment kinetics fitted either by a single exponent (red trace, <i>τ</i> = 66 ms, evaluated <i>D</i> = 3.5 µm²/s) or by a sum of two exponents (blue trace, <i>τ</i>₁ = 3.5 ms, <i>τ</i>₂ = 75 ms, <i>D</i> evaluated from <i>τ</i>₂ was 3.1 µm²/s; the amplitude of fast component was 0.105 of that for slow component). <i>D,</i> The relationship between the values of <i>D</i> prescribed in Monte-Carlo simulations and results obtained from fits as in <i>C</i> and <i>F.</i> The results were compared with identity line (red). The slopes of the best fits were: for Monte-Carlo simulations (as in <i>B–C</i>) 0.93±0.009 (circles), for the best monoexponential fit to calculated values 1.18±0.0006 (open triangles), for the slower mode of bi-exponential fit (as in <i>E–F</i>) 1.03±0.0004 (filled blue triangles).</p

Cumulative fusion involving post-fusion WPB.

<p>Selected images from a live cell epifluorescence video (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108093#pone.0108093.s001" target="_blank">Movie S1</a>) of an EGFP-CD63-expressing HUVEC stimulated with 100 µM histamine. Scale bar, 2 µm. <i>A</i>, Left, approximate positions of ROIs corresponding to two EGFP-CD63-labelled WPBs participating in the cumulative fusion. <i>A</i>, Right, images showing the change in shape of the first, dimmer WPB (ROI ‘1’) due to fusion. Arrow in frame 2 of <i>A</i> indicates a WPB that fuses to form initial rounded structure, indicated by arrows in frames 4–6 and frame 1 of <i>B</i>. Here and below the colour-coded bars above the images correspond to the marked time intervals in the graph <i>D</i>. <i>B</i>, Two rows of images showing the fusion of the second WPB ‘2’ with the rounded WPB ‘1’ indicated in <i>A.</i> The time of this second fusion was set to 0 s. Note the redistribution of fluorescence between the two structures. <i>C</i>, Loss of EGFP-CD63 fluorescence of the compound structure (arrow) upon fusion with the plasma membrane. <i>D</i>, The time course of the fluorescence changes within ROI ‘1’ (blue) and ROI ‘2’ (black) in control (black bar), during the cumulative fusion event (red bar) and after fusion with plasma membrane (blue bar). The average intensities of images were normalised to the beginning of experiment. The slow residual decline of fluorescence is due to uncompensated photobleaching of EGFP.</p

Post-fusion WPBs swell.

<p><i>A</i>, Example of the WPB shape change seen during stimulated (1 µM ionomycin) fusion in a HUVEC expressing EGFP-CD63. Scale bar, 2 µm. <i>B–C</i>, The distributions of the pre-fusion WPB lengths <i>L</i> (<i>B,</i> mean 1.56±0.06 µm) and of maximal radii of the resulting spheroid structures <i>R</i>₀ seen soon after fusion (<i>C,</i> mean 0.49±0.01 µm) for all WPBs measured when undergoing exocytosis (labelled with EGFP-CD63, <i>n</i> = 99 and with P-selectin-EGFP, <i>n = </i>41). <i>D,</i> The radius of rounded structure (<i>C</i>) plotted against the corresponding length of the parent WPB (<i>B</i>) in the same fusion event (points), double logarithmic scale. The solid line is the best power function fit to the dependence, <i>R</i>₀ = 0.384·<i>L</i>^0.573, the dashed line is the best √<i>L</i> fit: <i>R</i>₀ = 0.399·√<i>L</i>. The dotted line represents radius prediction for the model of rounding preserving the WPB surface, <i>R</i>₀ = 0.194·√<i>L,</i> the dash-dot line represents the constant-volume rounding model, <i>R</i>₀ = 0.162·<i>L</i>^1/3. In calculations the average diameter of WPBs was assumed to be 0.15 µm. <i>E</i>, the distribution of radii for all rounded fluorescent WPBs found persisting after stimulation and containing extracellular Alexa-647 (mean radius 0.61±0.02 µm, <i>n</i> = 150).</p