quasi-simultaneous electrochemical and electrophysiological measurements at the same sensor: probing...
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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).