Quasi-simultaneous Electrochemical and Electrophysiological Measurements at the Same Sensor: Probing the Chemical Environment and

Bioelectrical Activity of the Brain

Michael L. Heien1, Paul A. Garris4, Collin McKinney2, Regina M. Carelli3, R. Mark Wightman1

1Department of Chemistry, 2Electronics Design Facility, and 3Department of Psychology University of North Carolina, Chapel Hill, NC 27599-3290

4 Department of Biological Sciences, Illinois State University, Normal, IL 61790

IntroductionIntroduction

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 MicroelectrodesCylindrical 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

MethodsMethods

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 24 125 µA biphasic pulses (2 ms per phase), applied at 60 Hz.

Background Subtracted Cyclic VoltammetryBackground Subtracted Cyclic Voltammetry

300 V/s

-0.4 V

1.0 V

100 ms9.3 ms

Dopamine-o-quinone

NH2

OH

OH

NH2

O

O

+2H+

Dopamine

- 2e -

+ 2e -

-0.4 V1.0 V

-125 nA

125 nA

-0.4 V1.0 V

-125 nA

125 nA

-0.4 V1.0 V

-5 nA

5 nA

Analyzing DataAnalyzing Data

9.33 ms

Iout

0+1000 -400Eapp (mV vs Ag/AgCl)

CV

It

DA

1

A

3

2

Normalize to in vitro calibration3

2 Extract current at DA oxidation potential

1 Convert successive Iouts to pseudocolor

A Plot vs Eapp

col

Single Unit ElectrophysiologySingle Unit Electrophysiology

•Dopamine release can be detected using voltammetry

•Effect on postsynaptic activity can be measured

CV Period

100

µV

20 ms

Data CollectionData Collection

PC1 (Voltammetry)

TarHeel CV

PC2 (Electrophysiology)

Digitizer®

PCI-6052, NI

PCI-6711, NI

DACADC

DAC

Instrumentation

Breakout System

Preparation

Timing Signals

PCI-MIO-16E-4, NIADC

Neurolog®

Timing Signals

Voltammetric data was collected using in-house software. Electrophysiological data was collected using Digitizer®, and analyzed with Offline Sorter® (Plexon, inc.)

InstrumentationInstrumentation

Carbon FiberElectrode

Ag/AgClReference 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 SignalEffect of Holding Potential on Voltammetric Signal

0.1 -0.7-0.5-0.3-0.10.10.3 0.1

0

50

100

Holding Potential (V)

No

rmal

ized

Pea

kC

urr

ent

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 ScansElectrode Floating Between Scans

n = 4, 1 µM Dopamine injected, scanned from holding potential to 1 Volt

Contro

l

300

V/s

600

V/s

900

V/s

0

50

100

150

200

No

rmal

ized

Pea

kC

urr

ent

•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 Stability of In vivo In vivo Voltammetric SignalVoltammetric Signal

0 10 20 30 40 50 60

0

50

100

VoltammetryVoltammetry duringElectrophysiology

Time (min)

No

rmal

ized

Pea

kC

urr

ent

n = 3, 24 pulses, 60 Hz stimulation

Effect of Voltammetry on Neuron Firing RateEffect of Voltammetry on Neuron Firing Rate

36

Electrophysiology Only

0

25

50

75

36

Time (s)

Fir

ing

Fre

qu

en

cy

40

Switching, Scan Rate 300 V/s

0

25

50

75

40

Time (s)F

irin

g F

req

ue

nc

y

29

Switching, Scan Rate 600 V/s

0

25

50

29

Time (s)

Fir

ing

Fre

qu

en

cy

43

Switching, Scan Rate 900 V/s

0

25

50

75

43

Time (s)

Fir

ing

Fre

qu

en

cy

43

Switching, Scan Rate 1200 V/s

0

25

50

75

43

Time (s)

Fir

ing

Fre

qu

en

cy

44

Switching, Scan Rate 5000 V/s

0

25

50

75

44

Time (s)

Fir

ing

Fre

qu

en

cy

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 AccumbensNeuron Recorded in Nucleus Accumbens

Time (s)

Fre

quen

cy (

imp/

s)

1 ms

100

µV

Dopamine CVDopamine Signal

Dopamine Stimulation

Inhibition occurs simultaneously with DA release evoked by MFB stimulation

Unit Recorded

Behaviorally Evoked Responses in the Nucleus AccumbensBehaviorally Evoked Responses in the Nucleus Accumbens

Lever Press For Sucrose

-30 -15 0 15 300

0.2

0.4

0.60.8

11.2

1.4

Fre

quen

cy (

imp/

sec)

-0.61.4

-0.75

0.50

Dopamine CV

Time (s)

-5 0 5 10

Time (s)

150 n

M

Dopamine Signal

-30 -15 0 15 300

0.2

0.4

0.6

0.8

1

Dopamine Stimulation

Fre

quen

cy (

imp/

sec)

Time (s)

Fre

quen

cy (

imp/

s)

Fre

quen

cy (

imp/

s)

Inhibition occurs with both DA release evoked by MFB stimulation, and lever press for sucrose

Neuron Recorded in Nucleus AccumbensNeuron Recorded in Nucleus Accumbens

Excitation occurs after DA release evoked by MFB stimulation

Time (s)1 ms

100

µV

Fre

quen

cy (

imp/

s)

-5 0 5 10

Time (s)

300 n

M

Dopamine CVDopamine Signal

Dopamine Stimulation Unit Recorded

SummarySummary

• 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

AcknowledgementsAcknowledgements

• The authors would like to thank John Peterson, Joseph F. Cheer, and Mitchell F. Roitman.

• This work was supported NIDA DA14962 (RMC and RMW).