The MF approach performs well and leads into approaches to identify spike parameters.

Background: Quantal exocytosis of oxidizable neurotransmitters can be detected as spikes of amperometric current using electrochemical microelectrodes. Measurements of spike parameters indicate the maximal transmitter flux, flux duration, and amount of transmitter released from individual vesicles. Automated analysis algorithms need to reject spikes that overlap in time. In addition, many spikes are preceded by small amplitude “foot” signals, attributed to slow release of transmitter through a fusion pore. Accurate pre-spike baseline determination is essential for estimating fusion-pore duration and the amount of transmitter released through the fusion pore. New Method: We developed an estimation approach that is based on fitting a multi-exponential function to the data. Our previously described matched-filter algorithm is used to identify the sections of data to fit and provides seed values to facilitate convergence of the iterative fit. The new estimation algorithm includes overlap rejection, a two-step fitting procedure and a novel baseline estimation procedure. Results: Histograms of spike parameters demonstrate excellent agreement of the new approach with manually computed parameters. Comparison with Existing Methods: Parameter estimates generated using the new approach are closer to blind manual estimates than commonly used existing methods. The improved performance is due to better detection of valid spikes and rejection of overlapping spikes. Moreover, since the complete time course of the spike is fit to a function, more complete information about the spike time course is captured. Conclusions: The matched-filter seeded algorithm reliably rejects overlaps and estimates spike and foot signal parameters in a fully automated manner.

Electrochemical microelectrodes can detect single-vesicle release events as "spikes" of amperometric current. We developed a template based "matched-filter" approach that performs least squares fit of a library of templates to the data and identifies a spike when a detection criterion score given by the ratio of amplitude to the standard error exceeds a minimum threshold. This method outperformed existing approaches and detected >95% of true spikes for a mere 2% false positive rate as evidenced by receiver operating characteristic plots of sensitivity vs specificity. The next step is estimation of spike parameters like peak amplitude (Imax), half-maximal width (t50) and area under the curve (Q) which inform maximal flux, flux duration and charge respectively. Closely successive overlapping spikes are ambiguous to estimate as they may not decay back to baseline and should be rejected. Matched filter approach not only provided robust spike detection but also parameter seed values to reject overlapping spikes and also perform iterative curve fitting of spikes. The remaining well-separated spikes were iteratively fit in two phases, first by fitting rising and decaying phases separately and second by fitting the entire time course using seed values from the matched filter template parameters. Using curve-fit parameters, Imax, t50 and Q were calculated. Histograms of these parameters had bi-modal Gaussian distributions with centers and spreads within 12% and 4% of histograms created using manually analyzed data. The pre-spike baseline was estimated using a novel application of the matched-filter criterion scores and the estimation of pre-spike foot signal parameters such as charge (Qfoot) and duration (tfoot) yielded means, and medians within 10% of manually computed parameters.

depressed patients exhibit elevated catecholamine levels, perhaps suggesting common molecular targets or mechanisms. The serotonin transporter (SERT) mediates reuptake of serotonin (5HT) and is an important target for antidepressants. Notably, SERT is prominently expressed in adrenal chromaffin cells and 5HT co-localizes with epinephrine in chromaffin granules. However, the effects of 5HT/ SERT on chromaffin cell function remain unclear. Our data show that one role of SERT is to accumulate 5HT in chromaffin cells. The 5HT content of whole adrenal glands isolated from SERT knockout mice was reduced by~80% compared to wild-type littermates, but catecholamine content (~1000 fold higher than 5HT) was unaltered. We used carbon fiber amperometry to investigate the regulation of stimulus-secretion coupling by 5HT/ SERT. Cells were isolated from wild-type or SERT knockout mice, secretion evoked by 30mM KCl, and the number of amperometric spikes (vesicular fusion events) over 60s was quantified. Under control conditions (no 5HT) there was no difference in the number of spikes in wild-type Vs SERT knockout cells. However, extracellular 5HT (25nM-1mM) significantly reduced secretion in SERT knockout compared to wild-type cells. Similarly, when SERT was acutely blocked in wild-type cells using escitalopram (1mM), 5HT inhibited secretion. Escitalopram alone (no 5HT) had no effect, while 5HT alone produced a slight increase in secretion, although this was not statistically significant. Patch clamp experiments demonstrated that neither 5HT nor escitalopram altered Ca 2þ entry through voltage-gated calcium channels. Ongoing work will dissect the mechanisms underlying this complex regulation of chromaffin cells by 5HT/ SERT.

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