Evaluating the diffusion coefficient of dopamine at the cell surface during amperometric detection: disk vs ring microelectrodes

Abstract

During exocytosis, small quantities of neurotransmitters are released by the cell. These neurotransmitters can be detected quantitatively using electrochemical methods, principally with disk carbon fiber microelectrode amperometry. An exocytotic event then results in the recording of a current peak whose characteristic features are directly related to the mechanisms of exocytosis. We have compared two exocytotic peak populations obtained from PC12 cells with a disk carbon fiber microelectrode and with a pyrolyzed carbon ring microelectrode array, with a 500 nm ring thickness. The specific shape of the ring electrode allows for precise analysis of diffusion processes at the vicinity of the cell membrane. Peaks obtained with a ring microelectrode array show a distorted average shape, owing to increased diffusion pathways. This result has been used to evaluate the diffusion coefficient of dopamine at the surface of a cell, which is up to an order of magnitude smaller than that measured in free buffer. The lower rate of diffusion is discussed as resulting from interactions with the glycocalyx.

Figure 1

A) Average exocytotic spikes obtained at disk (blue line) and ring (red line) microelectrodes from a single typical amperometric trace; B) definition of the peak characteristics i_p, t_rise, t_1/2, t_fall and Q. The number of molecules released Q is represented by the gray area under the curve.

Figure 2

Histograms (30 bins, width: 0.005 s^1/2) for the half peak width (t_1/2)^1/2 (left) and the 75%–25% decrease time (t_fall)^1/2 (center) obtained for the ring (upper row) and disk (bottom row) geometries. The R² values obtained for the Gaussian fitting are shown on each panel. The histograms for Q^1/3 (50 bins, width: 20 molecules^1/3) obtained for the ring (upper row) and disk (bottom row) geometries are also shown on the right side of the figure.

Figure 3

Geometry of the system. Definition of the different parameters for A) a disk and B) a ring: r is the disk radius, r_out and r_in are respectively the outer and inner radii of the ring electrode. An additional distance, ε, at the edge of the carbon surface, is contributing to the formation of fast spikes and is drawn in red. The crown, i.e. the area generating diffusion distorted peaks, characterized by its thickness w, is shown in light blue (note that for the sake of clarity, the electrodes are here assumed to be circular, but they are actually elliptical because of the 45° beveling); C) scheme showing two vesicles fusing at the two limit locations of the cell surface described in the text (vesicles A and B produce slow and fast peaks, respectively) and the additional diffusion pathway Δx inducing the diffusional broadening at the edge of the electrode.

Figure 4

Simulation of the kinetics of exocytosis for different vesicle to center of the electrode distances, d, in the case of a ring electrode (I.D.: 1.5 µm, O.D.: 2.0 µm). A) Characteristic times t_1/2 or t_fall computed for different values of d (0, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1700, 1800, 1900, 2000, 2200, 2500, 2800, and 3000 nm). The light blue section indicates the position of the carbon ring. B) Plots of (Δx)² as a function of ${(\Delta \sqrt{t})}^2={(\sqrt{t}-\sqrt{t_0})}^2$, where t can be t_1/2 or t_fall (see text). The thin lines show the linear regressions for values of t_1/2 or t_fall associated to vesicles located into the crown.

Figure 5

TEM imaging of PC12 cells. A) The imaging of the membrane of a single PC12 cell shows the presence of a stained fuzzy layer (thickness: 17 ± 5 nm, mean ± SD from 5 random measurements at different sites of the image), in good agreement with TEM imaging of glycocalyx layer in monocytic cells (the white bar indicates 200 nm and 1 µm on the insert); B) TEM images showing the gap separating two PC12 cells, the thickness of the gap was measured as 24 ± 9 nm (mean ± SD from 5 random measurements at different sites of the image, the white bar indicates 200 nm). C) Variations of the calculated diffusion coefficient of dopamine at the surface of a PC12 cell D as a function of H, the width of the artificial synapse (average over the 4 values obtained for the ring and the disk electrodes, for t_1/2 and t_fall, ± SD). The blue areas indicate the reported thickness ranges for the extracellular matrix,^– the artificial synapses at a PC12 cell and at an artificial cell model. See Figure S3 in the Supporting Information for a plot of D for H values varying from 25 nm to 1 µm.