Figure 1
From: Bundles of Brain Microtubules Generate Electrical Oscillations
Experimental setup to study electrical oscillations of MT bundles. (a) Rat brain MT bundles obtained as reported in the Methods section (DIC x20). (b) Top Panel, DIC image of patch pipette approaching an MT bundle placed over a 2D MT sheet. Bottom Panel, Live immunochemical labeling of MTs by addition of both the anti-α ary antibodies to the preparation shown on Top. Please note that the loose patch connection between the MT bundle and the patch pipette allows backfilling of the pipette with both antibodies. (c) Top Panel, Schematics of the “loose-patch” clamp configuration to obtain electrical properties of MT bundles. The blue curved arrows from the pipette tip indicate leak currents that modify the amplitude of the holding potential applied from the amplifier. Bottom Panel, Schematic of the loose patch circuit applied to the MT surface. Resistances are shown for the pipette (Rpip), patch surface (RMT), seal (Rseal), and CMT represents the capacitive components for the MT surface (see Text for details). The holding potential (Command voltage, Vcmd) and tip potential (Vtip) are different, as expected for the loose patch configuration (See Methods). (d) Time series recording of a patched rat brain MT bundle under symmetrical KCl conditions (both pipette and bath), to which several holding (positive) potentials were applied as indicated in the Figure. Expanded tracings show the increase in amplitude and complexity of the spontaneous oscillations. (e) Time series of a recording held at 7 mV, showing changes in amplitude, spontaneous sudden death, and complete recovery of the oscillatory behavior in the absence of any changes in driving forces. (f) Top panel, Fourier power spectra obtained from (1) unfiltered current tracings from a free-floating pipette before attachment, (2) after attachment to the MT bundle, and (3) same sample after treatment with Taxol (10 μM). Bottom panel, 3D phase-space portraits from spectra shown on Top, indicating limit cycles for the attached sample under control conditions, and much reduced after Taxol treatment. Color code as in Top Panel. Delay time for first and second derivatives adopted for phase portraits was 1 ms.
