Quasi-simultaneous Electrochemical and Electrophysiological Measurements at the Same Sensor: Probing the Chemical Environment and Bioelectrical Activity of the Brain Michael L. Heien 1, Paul A. Garris 4, Collin McKinney 2, Regina M. Carelli 3, R. Mark Wightman 1 1 Department of Chemistry, 2 Electronics Design Facility, and 3 Department of Psychology University of North Carolina, Chapel Hill, NC Department of Biological Sciences, Illinois State University, Normal, IL 61790

Introduction Carbon fiber microelectrodes are frequently used to detect biogenic amines with in vivo voltammetry. An appealing application is to combine this approach with single-unit electrophysiology using the same sensor. Such a combination provides simultaneous information on the concentration of an easily oxidized neurotransmitter, such as dopamine, and its effect on postsynaptic neurons at exactly the same site. Prior work by Millar and colleagues has shown that fast scan cyclic voltammetry and single unit recording can be obtained using a single carbon fiber microelectrode [1]. We have developed instrumentation to accomplish these quasi- simultaneous measurements in freely moving animals. Voltammograms can be collected at a rate of 10 Hz, each lasting approximately 10 ms. In- between voltammograms, electrophysiological data are collected. Combined electrochemical and electrophysiological measurements are of similar quality to either measurement alone, although a small decrease in the voltammetric sensitivity is observed. [1] Williams, G. V., Millar, J. (1990). Neuroscience 39: 1-16.

Cylindrical Carbon Fiber Microelectrodes Micron dimensions – probe small areas Generates small currents – surrounding tissue remains undisturbed Low time constant – enhances time resolution and high speed applications are possible Low impedance (600 k  ) – Allows for high quality electrophysiological recordings (<10 µV RMS noise) Glass Seal Carbon Fiber 20 µm

Methods Triangle Waveform (Voltammetry) Electrophysiological Measurements A triangle waveform is applied to the carbon fiber microelectrode to make voltammetric measurements. Between voltammetric scans, electrophysiological measurements are made at the same electrode. A square pulse gates acquisition of voltammetric scans and electrophysiological data. A bipolar stimulating electrode was implanted in the MFB to evoke dopamine release. Stimulation parameters consisted of µA biphasic pulses (2 ms per phase), applied at 60 Hz.

Background Subtracted Cyclic Voltammetry 300 V/s -0.4 V 1.0 V 100 ms9.3 ms Dopamine-o-quinone NH 2 OH NH 2 O O +2H + Dopamine - 2e - + 2e V 1.0 V -125 nA 125 nA -0.4 V 1.0 V -125 nA 125 nA -0.4 V 1.0 V -5 nA 5 nA

Analyzing Data 9.33 ms I out E app (mV vs Ag/AgCl) CV It DA 1 A 3 2 Normalize to in vitro calibration 3 2 Extract current at DA oxidation potential 1 Convert successive I out s to pseudocolor A Plot vs E app col

Single Unit Electrophysiology Dopamine release can be detected using voltammetry Effect on postsynaptic activity can be measured CV Period 100 µV 20 ms

Data Collection PC1 (Voltammetry) TarHeel CV PC2 (Electrophysiology) Digitizer ® PCI-6052, NI PCI-6711, NI DAC ADC DAC Instrumentation Breakout System Preparation Timing Signals PCI-MIO-16E-4, NI ADC Neurolog ® Timing Signals Voltammetric data was collected using in-house software. Electrophysiological data was collected using Digitizer ®, and analyzed with Offline Sorter ® (Plexon, inc.)

Instrumentation Carbon Fiber Electrode Ag/AgCl Reference Electrode Preparation I/E Output (Voltammetry) Voltage Output (Electrophysiology) Ramp Signal Instrumentation has been miniaturized for work in freely moving animals. The headstage connects onto a stimulating electrode, while the electrode is loaded in a micromanipulator.

Effect of Holding Potential on Voltammetric Signal n = 6, 1 µM Dopamine, scanned from holding potential to 1 Volt at 300 V/s As the holding potential becomes more positive, the signal decreases due to potential dependent adsorption of dopamine DA Oxidation

Electrode Floating Between Scans n = 4, 1 µM Dopamine injected, scanned from holding potential to 1 Volt With electrophysiology between voltammetric scans, the potential of the electrode is allowed to float When the electrode’s potential is allowed to float, a decrease in voltammetric signal is observed One alternative is to increase the scan rate, because the signal is proportional to the scan rate

Stability of In vivo Voltammetric Signal n = 3, 24 pulses, 60 Hz stimulation

Effect of Voltammetry on Neuron Firing Rate Single unit recordings were made in the red nucleus while switching between voltammetry and electrophysiology. The scan rate employed was varied, which leads to larger currents at the microelectrode. Mean firing rates are shown as dashed lines

Neuron Recorded in Nucleus Accumbens Time (s) Frequency (imp/s) 1 ms 100 µV Dopamine CV Dopamine Signal Dopamine Stimulation Inhibition occurs simultaneously with DA release evoked by MFB stimulation Unit Recorded

Behaviorally Evoked Responses in the Nucleus Accumbens Inhibition occurs with both DA release evoked by MFB stimulation, and lever press for sucrose

Neuron Recorded in Nucleus Accumbens Excitation occurs after DA release evoked by MFB stimulation Time (s) 1 ms 100 µV Frequency (imp/s) Dopamine CV Dopamine Signal Dopamine StimulationUnit Recorded

Summary The instrumentation has been optimized for noise and bandpass requirements Quasi-simultaneous voltammetric measurements and electrophysiological measurements can be made with the same sensor A small decrease in the voltammetric sensitivity is observed, because of decreased adsorption The units recorded are not affected Quasi-simultaneous measurements can be made in freely moving rats

Acknowledgements The authors would like to thank John Peterson, Joseph F. Cheer, and Mitchell F. Roitman. This work was supported NIDA DA14962 (RMC and RMW).