Electrically evoking and electrochemically resolving quantal release on a microchip

Abstract

A microchip was applied to electrically depolarize rat pheochromocytoma (PC12) cells and to simultaneously detect exocytotic catecholamine release amperometrically. Results demonstrate exocytosis elicited by flowing cells through an electric field generated by a potentiostat circuit in a microchannel, as well as exocytosis triggered by application of an extracellular voltage pulse across. Electrical finite element model (FEM) analysis illustrated that larger cells experienced greater depolarizing excitation from the extracellular electric fields due to the smaller shunt path and higher resistance to current flow in the channel around the cell. Consistent with these simulations, data recorded from cell clusters and large cells exhibited increased release rates relative to data from the smaller cells. Overall, the system was capable of resolving single vesicle quantal release, in the zeptomole range, as well as the kinetics associated with the vesicle fusion process. Analysis of spike population statistics suggested detection of catecholamines from multiple release sites around the cells. The potential for such a device to be used in flow cytometry to evoke and detect exocytosis was demonstrated.

Fig. 1

(a) Optical micrographs of the MEMS microchip recording chamber with labeled: (i) fluidic channel (10 µm deep), (ii) two of the electrodes in the interdigitated array and (iii) a PC12 cell being pulled into the chamber. Also labeled are the working electrode (5 µm × 10 µm, WE) and counter electrode (CE) used in the potentiostat circuit. A separate Ag/AgCl reference electrode (not shown) is located further downstream. (b) The cell is isolated over the WE via a 5 µm constriction in the channel ensuring direct contact of the cell with the WE. Electrodes on either side of the cell are connected to high-impedance amplifiers for passive measurement of voltage across the cell (as shown).

Fig. 2

Quantal events. Example current trace recorded during amperometric (+700 mV vs Ag/AgCl) measurement of release from a cluster of six cells in the recording chamber. (INSET) (i) A typical recorded current spike corresponding to a quantal release event. (ii) In a subset of spikes, “foot” events (indicated by arrow) were detected consistent with the fusion pore formation hypothesis. (iii) Example of a “flicker” event which has been previously interpreted as resulting from closure of the fusion pore prior to full fusion of the synaptic vesicle with the plasma membrane.

Fig. 3

Quantal event analysis. Bin histograms were generated for a population of dexamethasone (5 µM)-treated PC12 cells (n = 209 cells, k = 25,463 spikes). (a) peak height (b) t_1/2 (c) cube root of quantal molar size. Fitting of the curves with double Gaussian functions suggested the detection of spikes from multiple release sites of the cells. Means (µ) and standard deviations (σ) for the Gaussian fits are shown.

Fig. 4

Correlates with cell size. Bars show the mean (±1 standard error) for various spike parameters as a function of cell size. Note: “# of spikes” (a) represents the number of quantal release events in the first 60 seconds of release. The four cell sizes (n = sample size) categories were: C = Clusters (two or more cells, n = 61), L = Large (n = 76), M = Medium (n = 34), S = Small (n = 10).

Fig. 5

FEM simulations to study effect of cell size. Images show the distributions of the voltages in the channel for a small cell (a) and large cell (b) for an applied WE voltage (DC, “δv” denoted voltage level in the channel). The color scale, indicating the fractional voltage distribution, highlights the electric field patterning technique (µ-domain voltage clamp). Relative to the extracellular fluid, the membrane impedance is very high resulting in the majority of the applied voltage dropping across the cell. The tighter fit of the larger cell creates more depolarization simply due to the restriction of the extracellular shunt path.

Fig. 6

Pulse evoked release. For small cells that were not sufficiently depolarized by potentiostat electric field, release could occasionally be triggered through the application of an extracellular voltage pulse. The graph above shows the working electrode current trace that shows onset of release following application of a voltage pulse (i).

Fig. 7

Exocytosis flow cytometry. Results for three separate cells flowing along the channel and crossing the WE in sequence are indicated at points marked i, ii, and iii respectively. Lower trace shows the working electrode current (red) and the upper trace shows the voltage drop (blue) recorded across the cell. Current spikes, corresponding to quantal release events, are detectable for all three cells, with a visible correlation between the spike intensity and the magnitude of (negative) change in ΔV across the cell.