Flexible software platform for fast-scan cyclic voltammetry data acquisition and analysis

Abstract

Over the last several decades, fast-scan cyclic voltammetry (FSCV) has proved to be a valuable analytical tool for the real-time measurement of neurotransmitter dynamics in vitro and in vivo. Indeed, FSCV has found application in a wide variety of disciplines including electrochemistry, neurobiology, and behavioral psychology. The maturation of FSCV as an in vivo technique led users to pose increasingly complex questions that require a more sophisticated experimental design. To accommodate recent and future advances in FSCV application, our lab has developed High Definition Cyclic Voltammetry (HDCV). HDCV is an electrochemical software suite that includes data acquisition and analysis programs. The data collection program delivers greater experimental flexibility and better user feedback through live displays. It supports experiments involving multiple electrodes with customized waveforms. It is compatible with transistor-transistor logic-based systems that are used for monitoring animal behavior, and it enables simultaneous recording of electrochemical and electrophysiological data. HDCV analysis streamlines data processing with superior filtering options, seamlessly manages behavioral events, and integrates chemometric processing. Furthermore, analysis is capable of handling single files collected over extended periods of time, allowing the user to consider biological events on both subsecond and multiminute time scales. Here we describe and demonstrate the utility of HDCV for in vivo experiments.

Figure 1

Results from in vivo recordings with a carbon-fiber electrode implanted in the nucleus accumbens of an anesthetized rat using a waveform designed for simultaneous detection of O₂ and dopamine. (A) Voltage waveform and data points used to produce the voltammograms. This scan is applied at 400 V/s every 100 ms. (B, C, D) Screen shots of the live, active color chart taken in three consecutive time intervals. Waveform data point number appears on the ordinate and time (s) is shown on the abscissa. Digital background subtraction has been set just before the present view. (B) Oxygen decreases are visible around data point 750. (C) An electrical stimulation evokes dopamine release (data points 160 and 450) and increases in oxygen. The oxygen change continues into the next 20 s time window (D).

Figure 2

Simultaneous electrochemistry/electrophysiology (echem/ephys) measurements. (A) Basic timing diagram during behavioral experiments. The state of the digital relay timing line controls when the potential of the electrodes floats (E_app dotted) and the waveform is applied (E_app solid). Behavioral events are recorded as digital transitions. (B) Example average (n = 30 trials) of dopamine concentration (grey trace) and average single unit firing rate (histogram) during ICSS recorded in the nucleus accumbens of an awake rat. The stimulating electrode in this study was implanted in the dopaminergic neurons of the ventral tegmental area. Data are aligned to the audiovisual cue. (C) Current response during waveform application. Voltage is applied to the electrode when the relay state is low while the recording window, defined by the CV application frequency, is denoted by the grey dashed lines. When the relay is in sync with the CV frequency clock (uncorrected, left) current spikes occur at the beginning and end of the voltage ramp. The CV frequency clock also triggers the relay in TarheelCV (middle), but the recording window is increased to add holding time before the voltage sweep. In HDCV (right) a line distinct from the CV frequency clock is configured to hold low 5 ms before and after the recording window.

Figure 3

Use of the 2D FFT filter. (A) Transformed view of the original and filtered (clipped) data. Signal intensity at each frequency is shown for the CV domain (ordinate) and the time domain (abscissa). Both axes are centered at 0 Hz and extend from −f_s/2 to f_s/2, where f_s is the sampling frequency. The −3 dB and −50 dB cutoffs are shown by the orange and black ellipses respectively. A Bessel roll-off is applied to smooth the transition between the −3 dB point and −50 dB point. Cutoff frequencies (time domain: 1.35 Hz, CV domain: 2 kHz) were chosen to resemble application of a 2 kHz low pass Bessel filter and an 8 point nearest neighbor smoothing kernel. (B) Comparison of filtering methodologies on data taken during in vivo norepinephrine release in the bed nucleus of the stria terminalis of an anesthetized rat. Release was evoked by electrical stimulation of the ventral noradrenergic bundle. The original unfiltered data is shown to the right. Stimulation onset is denoted at time 0. Norepinephrine concentration extracted by PCR is shown as a trace above the color plot. The CV below is taken from the time point indicated by the white dotted line. The middle panel shows this data after filtering with the 2 KHz low pass Bessel filter and smoothing kernel. The right panel shows the data after the 2D FFT filter with the parameters shown in part (A).

Figure 4

Continuous data analysis in HDCV. (A) Electrochemical recording of dopamine with the carbon-fiber electrode implanted in the nucleus accumbens of a freely-moving animal. Data is shown in 30 s file segments, as originally collected with the TarheelCV acquisition program. White audio noise begins at the bar above the color plots. Each file is background subtracted at the time indicated by the yellow dotted line. (B) Data from part (A) concatenated into a continuous HDCV file through a data convertor program. Unlike TarheelCV, HDCV reads only the requested portion of the data file at a single time permitting long periods of data to be analyzed with a single background subtraction time point (yellow dotted line). (C) Background-subtracted CVs taken from the 30 s and continuous files at the same time point (white dotted lines in A and B). (D) PCR of the continuous data reveals a decrease in dopamine concentration over time. Based on the residual analysis (inset) the concentrations in the shaded box are statistically unreliable.

Figure 5

Time bin analysis procedure. (A) A five minute view of continuous data collected during ICSS. HDCV displays both electrochemical (color plot) and digital input data simultaneously on its main screen. (B) A time bin extracted from the data in (A). The time bin is aligned to the falling edge of line four, which indicates the presentation of an audiovisual cue. Digital transitions corresponding to lever out, lever press and stimulation events also occur within this time frame. (C) Progression plot showing dopamine concentration (MM) versus time for 40 consecutive ICSS trials aligned to the cue. (D) Single and average (n = 40 trials) time bin data extracted with the training set shown in (C). Dopamine concentration is shown as a trace above each color plot. The identity and timing of the alignment event is indicated by the dotted vertical lines.