Increasing the sensitivity of microelectrodes
Christopher B. Jacobs, Andrew M. Strand, B. Jill Venton*
Department of Chemistry, University of Virginia, Charlottesville, VA 22904
(*bjv2n@virginia.edu)
Introduction
Carbon-fiber microelectrodes have been used for in vivo measurements of
neurotransmitters. Our lab is exploring a few different approaches to increasing
the sensitivity of these electrodes. The first approach is to modify the surface of
electrodes with carbon nanotubes. Carbon nanotube treatments of electrodes
have been used to increase sensitivity, promote electron transfer, and reduce
fouling. Most methods have focused on nanotube coatings of large electrodes
and slower electrochemical techniques that are not conducive to measurements in
vivo. We have investigated carbon-fiber microelectrodes modified with singlewalled carbon nanotubes for co-detection of dopamine and serotonin in vivo.
A second approach to increasing sensitivity is flame etching the microelectrodes.
This results in electrodes with a 1-2 µm diameter as opposed to normal fibers
with 7 µm diameters. These small electrodes have a higher signal per unit area
and have higher S/N ratios than normal microelectrodes. The flame etched
electrodes should cause less tissue damage and facilitate measurements in small
places or small model organisms.
Methods
Carbon nanotube modified microelectrodes. Single-walled carbon nanotubes
(SWCNT), prepared by the arc method, were purchased from CarboLex
(Lexington, KY). They were purified and oxidized by stirring in a 2 M HNO3
solution for 24 hours. A 0.5 mg/ml suspension of oxidized nanotubes in 2.5 %
Nafion (diluted with isopropanol from the 5 % stock solution of Nafion, Sigma)
was sonicated for 30 min in a water bath sonicator to suspend the nanotubes.
Carbon-fiber microelectrodes were dipped for 1 s in the nanotube suspension
solution and then immediately dipped for 30 s in isopropanol as a rinse to remove
loose nanotubes. Electrodes were allowed to air dry at least 10 min before they
were tested.
Etched electrodes. Carbon-fiber microelectrodes were fabricated from T-650
fibers (7 µm diameter, Cytec Engineering Materials, West Patterson, NJ) The
fiber extending from the glass was cut with a scalpel under the microscope so
that it extended 100 to 150 µm from the glass seal. To flame etch, the electrodes
were held in a butane flame (Lenk butane torch, Kinston, NC) for about 3
seconds. The flame temperature was approximately 2100ºF and the electrode
was held just on the edge of the blue part of the flame. Positioning was
important, as placing the electrode in the hottest part of the flame tended to
destroy the glass seal. As the very end of the tip became red, the electrode was
rotated in order to ensure even etching on all sides. Other electrodes were
electrochemically etched in a solution of 0.5 mM K2Cr2O7 in 5 M H2SO4 [1].
This solution was suspended in a small platinum loop and the electrode
positioned in the drop by a micromanipulator. A 6 V, 60 Hz sine wave was
applied between the electrode and platinum wire for 20-30 s to etch. All
electrodes were soaked for at least 10 min. in isopropanol immediately before
use.
Results and Discussion
Carbon nanotube modified microelectrodes. In this study, we investigated
carbon-fiber microelectrodes modified with a thin film of single-walled carbon
nanotubes for co-detection of dopamine and serotonin in vivo [2]. Applying a
thin layer of carbon nanotubes resulted in a 2.5-fold increase in sensitivity for
detection of dopamine and serotonin when using fast-scan cyclic voltammetry.
However, electrocatalytic effects of nanotubes were not apparent at fast scan
rates. Nanotube-modified microelectrodes showed significantly less fouling after
exposure to serotonin than bare electrodes. The nanotube-modified electrodes
were used to monitor stimulated dopamine and serotonin changes simultaneously
in the anesthetized rat after administration of a serotonin synthetic precursor.
These studies show that nanotube coated microelectrodes can be used with fast
scanning techniques and are advantageous for in vivo measurements of
neurotransmitters because of greater sensitivity and resistance to fouling. We are
continuing this work by investigating other solvents for suspending the carbon
nanotubes. Preliminary evidence shows that solvents such as dimethylformamide
and dimethylsulfoxide are good for suspension. We are also investigating
making fibers out of carbon nanotubes to reduce the interference effects of the
carbon fiber and more accurately assess the electrochemical properties of the
nanotubes.
Etched electrodes. Flame etching results in electrodes with a 1-2 µm diameter as
opposed to normal fibers with 7 µm diameters. These small electrodes have a
higher signal per unit area and have higher S/N ratios for 1 µM dopamine than
normal carbon-fiber microelectrodes or electrochemically etched electrodes [3].
Therefore, the increased sensitivity is not just a property of size. The flameetched surfaces had nanometer-scale surface features that were not observed on
the other electrodes and exhibited increased sensitivity for other electroactive
compounds found in the brain, including ascorbic acid, DOPAC, and serotonin.
Faster kinetics and a faster response to a step change in dopamine were also
observed, when the applied waveform was -0.4 to 1.0 V and back at 400 V/s.
The sensitivity of the flame-etched electrodes was enhanced by overoxidizing the
surface. The flame-etched electrodes were used to detect dopamine release in
anesthetized rats after a single stimulation pulse. The flame etched electrodes
should cause less tissue damage and facilitate measurements in small places. For
example, ongoing efforts in our laboratory are being made to measure
neurotransmitters in fruit fly nerve cords that are only 50 µm wide.
A. Background
B. 1 µM Dopamine
1000 nA
12 nA
normal
flame-etched
electrochem-etched
1.0 V -0.5 V
-0.5 V
-1000 nA
1.0 V
-12 nA
Figure 1. Cyclic voltammograms from etched electrodes. Background charging
currents for 125 µm-long normal (solid line), flame-etched (red dashed line), and
electrochemically etched (blue dotted line) carbon-fiber microelectrodes. Electrodes
were scanned from -0.4 to 1.0 V and back at 400 V/s every 100 ms. B.) Backgroundsubtracted cyclic voltammograms for 1 µM dopamine. Colors are the same as in panel A.
References
1.
2.
3.
Kawagoe KT, Jankowski JA, Wightman RM (1991) Etched carbon-fiber
electrodes as amperometric detectors of catecholamine secretion from isolated
biological cell. Anal Chem 63:1589-1594.
Swamy BEK, Venton BJ (2007) Carbon nanotube-modified microelectrodes for
simultaneous detection of dopamine and serotonin in vivo. Analyst 132:876-884.
Strand AM, Venton BJ (2008) Flame etching enhances the sensitivity of carbonfiber microelectrodes. Anal Chem 80:3708-3715.
Electrochemical imaging of dopamine release on single PC12 cells
Bo Zhang1, Michael Heien2, Kelly Adams2, Andrew G. Ewing2,3*
1
Department of Chemistry, University of Washington, Seattle WA 98195; 2Department of
Chemistry, the Pennsylvania State University, University Park, PA 16802; 3Department
of Chemistry, Göteborg University, Kemivägen 10, SE-41296, Göteborg, Sweden
(*andrew.ewing@chem.gu.se)
Introduction
Electrochemical methods utilizing carbon-fiber microelectrodes have been
extremely useful in the detection of easily oxidizable neurochemicals (e.g.,
dopamine, epinephrine, 5-hydroxytryptamine, and histamine) from single
cells.[1,2] Experiments using a single carbon-fiber microelectrode provide
valuable information such as chemical identity, amount of the neurotransmitter
released, event frequency, and kinetic information relating to fusion pore
opening.[3] However, spatial heterogeneity of exocytotic events is difficult to
study.
We report the fabrication and characterization of carbon microelectrode arrays
(MEAs) and their application to spatially and temporally resolve neurotransmitter
release from single pheochromocytoma (PC12) cells. The carbon MEAs are
composed of individually addressable 2.5-μm-radius microdisks embedded in
glass. The fabrication involves pulling a multibarrel glass capillary containing a
single carbon fiber in each barrel into a sharp tip, followed by beveling the
electrode tip to form an array (10-20μm) of carbon microdisks. The carbon
MEAs have been characterized using scanning electron microscopy, steady-state
and fast-scan voltammetry, and numerical simulations. Both amperometry and
fast-scan cyclic voltammetry (FSCV) have been applied to electrochemically
image single PC12 cells. Amperometric results show that subcellular
heterogeneity in single-cell exocytosis can be electrochemically detected with
MEAs. FSCV results reveals that there is strong cross-talk on adjacent
microelectrodes. Temporal resolutions in both amperometry and FSCV
experiments have been discussed. These ultrasmall electrochemical probes are
suitable for detecting fast chemical events in tight spaces, as well as for
developing multifunctional electrochemical microsensors.
Methods
Fabrication of carbon microelectrode arrays. The fabrication of seven carbon
MEAs involved insertion of individual carbon fiber into a seven-barrel glass
capillary followed by pulling the glass capillary on a commercial glass puller.
Detailed information can be found in reference 4.
Electrochemical recording. Electrochemical recordings on single PC12 cells
were made on an inverted microscope (CK30, Olympus) placed in a Faraday
cage. Four bipotentiostats (EI-400, Ensman) were used to perform both the
amperometry and FSCV experiments on carbon MEAs. In amperometry
recording, the bipotentiostats were interfaced to a PC computer through a multichannel data acquisition system (Digidata 1440A, Molecular Devices). FSCV
experiments were performed using a home-built FSCV system.
Finite-element simulation. The temporal resolutions in both amperometric and
FSCV recordings, the cross-talk in voltammetric experiments were simulated
using Comsol Multiphysics® 3.2 software.
Results and Discussion
Electron microscopic characterization showed that these micro-sensor arrays
were on the order of 20 μm and were geometrically well defined, as shown in
Figure 1b. Figure 1c reveals that subcellular heterogeneity in exocytosis was
shown with 5-μm resolution in our amperometric imaging. Concurrent exocytotic
events under different microelectrodes were also resolved using MEAs. Figure 2
shows 15s period of a FSCV imaging on a single PC12 cell. Co-detection of
individual exocytotic events on adjacent microelectrodes can be clearly seen with
FSCV imaging. Temporal resolutions in both amperometry and FSCV, the crosstalk in voltammetric experiments were simulated using finite-element
simulations.
Our results show that carbon-fiber based microelectrode-arrays are suitable for
electrochemical imaging of fast exocytotic events at single cells.
(c)
Figure 1. (a) Schematic diagram and (b) SEM images of carbon microelectrode arrays
composed of two, three, and seven individually addressable electrodes. Each electrode tip
measures ~5 µm in diameter, (c) Amperometric traces of exocytotic release from a PC12
cell recorded using a microelectrode-array. Thick black lines along the time axis indicate
exposure to high potassium stimuli (100 mM, 5-s pulse every 45 s).
Figure 2. A 15 second time period of FSCV imaging on a single PC12 cell using a seven
carbon MEA, the scan rate was 1500 V/s.
References
1.
2.
3.
4.
Wightman RM, Jankowski JA, Kennedy RT, Kawagoe KT, Schroeder TJ,
Leszczyszyn DJ, Near JA, Diliberto EJ Jr, Viveros OH (1991) Temporally
resolved catecholamine spikes correspond to single vesicle release from
individual chromaffin cells. Proc. Natl. Acad. Sci. USA 88:10754-10758.
Chen TK, Luo G, Ewing AG (1994) Amperometric Monitoring of Stimulated
Catecholamine Release from Rat Pheochromocytoma (PC12) Cells at the
Zeptomole Level, Anal. Chem. 66:3031-3035.
Mosharov E, Sulzer D (2005) Detailed algorithms for detection and analysis of
exocytic events recorded by amperometry. Nature Methods, 2:651-658.
Zhang B, Adams, KL, Luber SJ, Eves DJ, Heien ML, Ewing AG (2008)
Spatially and Temporally Resolved Single-Cell Exocytosis Utilizing
Individually- Addressable Carbon Microelectrode Arrays.
Anal. Chem.
80:1394-1400.
In vivo real time measurements of dopamine neurotransmission using
microelectrode arrays with constant potential amperometry
M Lundblad*, P Huettl, F Pomerleau, G A Gerhardt
Morris K. Udall Parkinson’s Disease Research Center of Excellence, Center for
Microelectrode Technology, Department of Anatomy and Neurobiology, University of
Kentucky
(*kmlund3@uky.edu)
Introduction
To study dopaminergic neurotransmission in vivo, the recording technique should
have (i) high temporal resolution to study fast changes in dopamine (DA) levels
during release and reuptake, (ii) high spatial resolution to enable measurements
from small structures and subdivisions of larger dopaminergic target structures
such as the striatum, and (iii) low limit of detection to detect basal/resting DA
levels in the area of interest. Presently, microdialysis is the most commonly used
technique to measure DA in the brain. Even with improvements of temporal
resolution in recent years (up to 1 sample/20s) the nature of the sampling
procedure does not allow for very high temporal resolution. To further increase
temporal and spatial resolution, other techniques such as fast scan cyclic
voltammetry and amperometry coupled with microelectrodes can be employed.
In the present studies we have developed a new technique involving the use of
microelectrode arrays (MEAs) coupled with amperometric recordings to measure
resting levels of extracellular DA in the striatum of anesthetized or awake rats.
Methods
Microelectrode arrays (S2 15 μm x 333 μm recording sites) were used for all
amperometric recordings. The Pt sites were first coated with Nafion® to repel
anionic interferents. Subsequently, m-phenylenediamine (m-PD) was
electroplated onto one site of each pair of Pt-sites. The m-PD creates a size
exclusion layer which blocks DA and other large molecules from reaching the Pt
recording site. DA was oxidized (+0.35 V vs. Ag/AgCl) on the Pt sites and
generated a current, which was amplified and digitized using the FAST-16 mkII
recording system. Each electrode was individually calibrated against standard
DA and ascorbic acid increments to verify linearity (only MEAs with r2>0.990
were used) and selectivity (selectivity of all electrodes were >300:1 for DA
versus ascorbic acid).
Animal surgery. Depletion of DA in the striatum was induced by infusion of 6OHDA into medial forebrain bundle as previously described [1].
Recordings from anaesthetized animals. Animals were anaesthetized by i.p.
injection of 1.25 g/kg of urethane. MEAs were placed in the dorsal striatum using
these coordinates: from bregma AP:+1.0mm, ML:-2.5mm, DV:-3.5 from the
dural surface.
Basal DA recordings: The signal (current) from the m-PD coated site was
subtracted from the Nafion-coated site signal in each pair of recording sites. This
results in one signal from each pair of recording sites, referred to here as the selfreferenced signal.
The intrinsic background current of the recordings sites in vivo, that was not
possible to filter out by the self-referencing method, was discovered in early in
vivo recordings. To adjust for this intrinsic background, a stable baseline
recording (self-referenced signal) was obtained from cortex (that is practically
devoid of DA) in each animal. This signal was subsequently subtracted from the
baseline striatal recordings in the same animal to produce a selective basal DA
signal. Limit of detection for this procedure was <1 nM for all recordings.
Pharmacological treatment experiments: Normal animals were anaesthetized
with urethane (1.25 g/kg). When a stable DA baseline was reached, the animals
were injected with either amphetamine or cocaine.
Recordings from freely moving animals: Implantation of the MEA was made as
previously described [2]. Rats were anaesthetized using isoflurane inhalation
(1.5-4%). An MEA prepared for DA measurement was chronically implanted in
the striatum and kept in place using dental cement.
Results and Discussion
KCl-evoked DA release was nearly abolished after the extensive unilateral 6OHDA lesions (Fig. 1A and 1C). The temporal profile and amplitude of the DA
release as measured with the MEAs in normal striatum was very similar to those
previously recorded using single carbon fiber electrodes [3]. More importantly,
basal level recordings using the DA selective MEA with self-referencing and
cortex baseline subtraction showed a dramatically reduced extracellular
concentration of DA in the striatum of 6-OHDA lesioned rats (Fig. 1B).
Figure 1. KCl-evoked and basal concentration of DA in anaesthetized rats. KCl-evoked DA
release in the DA depleted striatum (red line) and the unlesioned striatum (black line) in the same
animal (A). The basal concentration in striatum of 6-OHDA lesioned and sham lesioned animals
(one way ANOVA *p<0.001) (B). HPLC analysis of striatal DA content in the normal, sham
lesioned and 6-OHDA lesioned animals used for amperometric recordings (one way ANOVA
*p<0.001, tukey post hoc test)(C).
To further test the sensitivity of the technique, low doses of agents known to
affect extracellular levels of DA were injected i.p. in anaesthetized animals. As
expected, a low dose of cocaine (10 mg/kg) caused an increase in extracellular
DA in rat striatum (Figure 2A). The temporal profile showed a slow and stable
increase of extracellular DA. In contrast, after injection of d-amphetamine (2.0
mg/kg i.p.) the temporal profile showed oscillations of extracellular DA
concentration (Fig. 2 B). While both drugs increased basal DA concentration, the
high resolution temporal profile that can be produced using this technique
showed distinct characteristics of the response to the different drugs.
Figure 2. Self-referenced signal, after cortex baseline subtraction showing basal DA
concentration after i.p. injection of cocaine 10 mg/kg (A) and d-amphetamine (2.0
mg/kg) (B).
To eliminate any potential effect of anesthesia, the DA selective MEAs were
modified and implanted for freely moving recordings. To verify the effects of damphetamine, the same dose (2.0 mg/kg) was given 1 and 3 days after
implantation of the MEA. The freely moving recordings produced similar
temporal profiles as those recorded in anaesthetized animals. The oscillations
however seemed faster and had higher amplitudes in awake rats (preliminary
data).
Figure 3. Self referenced amperometric signal from awake, freely moving rat before (A)
and after (B) 2.0 mg/kg d-amphetamine i.p. 3 days after MEA implantation.
In conclusion, these results showed that we can selectively measure DA in vivo
using high speed amperometry with the MEAs. Both evoked release and basal
levels of DA can be studied using this technology and the sensitivity is sufficient
to monitor the dramatic effects such as a nearly complete reduction of releasable
DA and extracellular DA using 6-OHDA lesions and subtle changes such as
pharmacological treatment with low dose amphetamine or cocaine. Furthermore,
this technology can be used in awake animals with the same sensitivity and
selectivity for at least 3 days.
References
1.
2.
3.
Lundblad M, Andersson M, Winkler C, Kirik D, Wierup N, Cenci MA (2002)
Pharmacological validation of behavioural measures of akinesia and dyskinesia
in a rat model of Parkinson's disease. Eur J Neurosci 15:120-132.
Rutherford EC, Pomerleau F, Huettl P, Strömberg I, Gerhardt GA (2007)
Chronic second-by-second measures of L-glutamate in the central nervous
system of freely moving rats. J Neurochem 102:712-722.
Hebert MA, Van Horne CG, Hoffer BJ, Gerhardt GA (1996) Functional effects
of GDNF in normal rat striatum: presynaptic studies using in vivo
electrochemistry and microdialysis. J Pharmacol Exp Ther 279:1181-1190.
Simultaneous measurements of rapid dopamine release and
accumbens cell firing during cocaine self-administration
Regina M. Carelli1*, Catarina A. Owesson-White1,2, Jennifer Ariansen2,
Garret D. Stuber3, Joseph F. Cheer2 and R. Mark Wightman2
1
Department of Psychology, 2Department of Chemistry, 3 Curriculum in Neurobiology,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
(*rcarelli@unc.edu)
Introduction
Mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA)
to the nucleus accumbens (NAc) are part of a complex circuit mediating rewarddirected behaviors. Electrophysiological recordings in behaving rodents have
revealed that distinct populations of NAc neurons exhibit changes in cell firing
(increases and/or decreases) within seconds of lever press responding for natural
(food/water), drug or ICS reward ([1, 2]). These discharge patterns encode the
important features of goal-directed behaviors, as well as environmental cues
associated with reward. Using fast scan cyclic voltammetry (FSCV) in awake
animals, we have also measured rapid (subsecond) dopamine release events that
are temporally analogous to data obtained from electrophysiological recordings
during goal-directed behaviors ([3, 4]). However, definitive statements about
the precise relationship between specific types of cellular discharges and
dopamine could not be determined in those studies since measurements were
completed in different animals. Moreover, it is not known whether
differences exist in rapid dopamine signaling and NAc activity across the two
major subregions of the NAc, the core and shell. Here, two experiments were
completed to compare the temporal characteristics of rapid dopamine release
events within the NAc core versus shell using FSCV (Exp. 1), and to determine
the relationship between phasic dopamine and NAc cell firing (Exp.2) using a
combined electrophysiology/electrochemistry technique.
Methods
Male, Sprague–Dawley rats (Harlan), approximately 90–120 days old were used
as subjects. All animals were surgically prepared for cocaine self-administration
and trained to press a lever for intravenous cocaine (0.33 mg/inf, 6s) during daily
2 h sessions. For experiment 1 (FSCV only) rats were prepared for
electrochemical recording in the NAc core and shell as described previously [5].
A fresh carbon-fiber microelectrode was lowered into the NAc core or shell and
its potential was held at –0.4V versus an Ag/AgCl reference electrode.
Voltammetric recordings were made every 100ms by application of a triangular
waveform in which the potential was driven to +1.3V and back at a rate of
400V/s. In the presence of dopamine, the output current consists of a negative
(oxidation) peak followed by a positive (reduction) peak. When this is plotted
against the input potential, a cyclic voltammogram (CV) is produced, which
allows for chemical identification of dopamine and other electroactive species.
Following electrode equilibration, dopamine was electrically evoked by
stimulating the VTA to ensure that the electrode was positioned close to release
sites. After in vivo recordings, electrically evoked dopamine release was used to
identify naturally occurring dopamine transients using principal component
regression and data were aligned to lever pressing. For Experiment 2, a combined
electrophysiological and electrochemical recording procedure was used as
previously described ([6]). Briefly, FSCV was used as described above but
voltammograms were collected at 5 Hz with each scan taking 10 ms; another 10
ms around the triangular waveform was excluded to allow for switching between
electrophysiological and FSCV recordings. A solid-state relay in the headstage
alternated between a current amplifier for voltammetric scans and a voltage
follower for unit recording. The unit recordings were collected in 180 ms
intervals. Waveforms were discriminated using principal component analysis in
Offline Sorter (Plexon Inc, Dallas, TX). Neuronal cell firing was classified into
one of three well defined types of patterned discharges relative to the reinforced
response for cocaine as described previously ([1]).
Results and Discussion
In Experiment 1, the temporal characteristics of rapid dopamine release events
relative to the reinforced response for cocaine during self-administration was
compared across the NAc core and shell. Consistent with our prior reports [3]
dopaminergic signals at the lever press for intravenous cocaine in the core had
two distinct components; an initial increase in dopamine within seconds before
the response followed by a larger more sustained increase following response
completion. Although increases in dopamine concentration were also observed
before and following the response at shell locations, the dual increases included a
larger pre-response increase, and a less-synchronized post-response component
than that observed in the core. Thus, dopamine signaling in the shell relative to
the reinforced response for cocaine was observed as in the core, but appeared to
be of longer duration and was less synchronized to the response than that
observed in the core.
In Experiment 2, we determined whether locations at which phasic changes in
dopamine release are present are the same locations at which NAc neurons
exhibit patterned discharges. A total of 75 NAc neurons (34 cells in the core; 41
cells in the shell) were recorded in combination with voltammetric measurements
of phasic dopamine release. All NAc cells were recorded at places where
electrically-evoked dopamine release was elicited (i.e., in dopamine rich
regions). Of the 75 NAc cells, 29 neurons (39%) exhibited one of three types of
patterned discharges within seconds of the reinforced response for cocaine
previously described [1]. A major finding of this study was that locations where
phasically active cells were recorded were coincident with sites at which rapid
dopamine release was observed in the core and shell. Figure 1 shows an example
of a single NAc shell neuron that exhibited patterned cell firing; phasic dopamine
release was observed relative to the response at this location. Moreover, of the
75 NAc neurons, 46 cells (61%) showed no change in cell firing relative to the
cocaine-reinforced response (termed nonphasic or NP cells). Remarkably, at all
locations at which NP cells were recorded, no significant changes in rapid
dopamine release was observed relative to the response. These results support the
view that the NAc is a functionally heterogeneous structure and that rapid
dopamine release may play a critical role in the activation of specific subsets of
NAc neurons that encode cocaine-directed behaviors.
110 µV
Mean Firing Rate (Hz)
Figure 1: Simultaneous electrophysiological recording and electrochemical
measurements in the NAc shell reveal patterned cell firing and phasic dopamine
release relative to the cocaine-reinforced response. Top: Color plot averaged across
‐ 0.4
all self-administration trials of cyclic
voltammetric data shows an increase in
dopamine concentration immediately
before the response, with a second peak
+ 1.3
increase in dopamine approximately 4 s
following the response. The dopamine
concentration relative to the lever press
‐ 0.4
was confirmed with principal component
regression
(bottom,
blue
trace
‐ 0.7
1.0 nA
superimposed on the PEH). Bottom: PEH
and corresponding raster display show the
activity of a single simultaneously
14
recorded NAc neuron (waveform to the
right). In this case, the neuron showed an
25 nM
increase in cell firing that began within 1 s
7
following response completion, and
0.7 ms
maintained a sustained increase in activity
during the 10 s post response period.
-10
-5
R
5 s ec 10
References
1.
2.
3.
4.
5.
6.
Carelli RM, Ijames SG, Crumling AJ (2000) Evidence that separate neural
circuits in the nucleus accumbens encode cocaine versus "natural" (water and
food) reward. J Neurosci 20:4255-4266.
Cheer JF et al (2007) Coordinated accumbal dopamine release and neural
activity drive goal-directed behavior. Neuron 54:237-244.
Phillips PE et al (2003) Subsecond dopamine release promotes cocaine seeking.
Nature 422:614-618.
Roitman MF et al (2004) Dopamine operates as a subsecond modulator of food
seeking. J Neurosci 24:1265-1271.
Day JJ et al (2007) Associative learning mediates dynamic shifts in dopamine
signaling in the nucleus accumbens. Nat Neurosci 10:1020-1028.
Cheer JF et al (2005) Simultaneous dopamine and single-unit recordings reveal
accumbens GABAergic responses: implications for intracranial self-stimulation.
Proc Natl Acad Sci U S A 102:19150-19155.
Long-term, chronic microsensors for multisite, real-time dopamine
detection in behaving animals
Stefan G. Sandberg, Jeremy J. Clark, Scott B. Evans, Jerylin O. Gan,
Matthew J. Wanat, Paul E. M. Phillips*
Department of Psychiatry & Behavioral Sciences, and Department of Pharmacology,
University of Washington, Seattle, WA 98195-6560, USA
(*pemp@u.washington.edu)
Introduction
Phasic burst firing of midbrain dopaminergic neurons is thought to transiently
increase extracellular concentrations of dopamine ([DA]EC) [1] and has been
correlated with associative and procedural learning [2,3]. The most suitable
technique for measuring fast transient changes in [DA]EC of freely moving rats is
in vivo voltammetry at the carbon fiber microelectrode (CFM). However, one
drawback of in vivo voltammetry has been the inability to perform within animal
comparisons, due to the necessity for session-to-session re-implantation of a
fresh CFM [4]. This, in turn, makes obtained DA measurements difficult to
compare between sessions. Within animal comparisons are especially critical
when investigating dopamine’s role in behavioral changes over time, as in
associative learning tasks, for example during acquisition of a pavlovian
conditioned approach task (Clark et al., this volume). As a solution to this
problem we have developed a new CFM for chronic implantation into any
terminal field of dopaminergic neurons.
Methods
Chronic CFM’s were constructed from fused silica coated with a biocompatible
polyamide layer. Male Sprague-Dawley rats (300-400g) were anesthetized with
isofluorane (2-3%) and immobilized in a stereotaxic apparatus. CFM was
implanted according to flat skull coordinates, referenced to bregma at AP: +1.3,
ML:+1.3, and referenced from top of brain DV:-7.0-7.2 (all measurements in
mm). Reference electrode (Ag/AgCl) was placed in the contralateral hemisphere.
All electrodes were secured with dental cement. Animals were allowed to recover
for 1 month before any data collection was made. In vivo voltemmetry was
utilized for detection of electrically evoked (60Hz, 24p, 120 μA) or behaviorally
evoked dopamine (delivery of 1 unexpected food pellet) at the chronic CFM.
Results and Discussion
Here we have successfully implanted CFM for simultaneous DA measurements
in multiple brain areas of the rat. This experiment is the first of its kind and
further highlights the potential of this electrode. Moreover, due to its small size,
voltammetric approaches are now feasible in smaller species, such as mice and
birds. This confers additional advantages for elucidating the interactions between
gene regulation, neurotransmission and behavior, since mice are especially
suitable for genetic manipulations. Similar to CFM’s for acute implantation, the
chronic CFM is selective and sensitive to electrically and behaviorally evoked
release (Figure 1). This is illustrated by pharmacological inactivation with
baclofen of ventral tegmental area dopaminergic neurons leading to a significant
reduction of otherwise consistent DA release in response to delivery of an
unexpected food pellet. To further characterize the DA sensitivity of the chronic
CFM’s in vitro pre and post calibration will be employed.
Surgery
1st Month
2 Months
2nd Month
4th Month
1000 nM
100 nM
2s
2s
Figure 1. In vivo detection of dopamine release at chronic carbon-fiber
microelectrode. Left panel, Electrically evoked dopamine (60 Hz, 24 p, 120 μA)
measured at surgery and 2 months post surgery. Solid line below trace denotes electrical
stimulation application. Right panel, Behaviorally evoked dopamine elicited by delivery
of unexpected food pellet at 1, 2 and 4 months post surgery. Arrow below trace denotes
time of pellet delivery. Scale bars in both figures represents time (abscissa) and
concentration of dopamine (ordinate).
These data demonstrate the versatility of the chronic CFM as DA release can be
measured on a trial-to-trial basis. This, in addition to being able to measure DA
release in regionally specific terminal domains offers a solution to not knowing
the projection of dopaminergic neurons, as seen in electrophysiological
studies[5]. This electrode combines the advantages of microdialysis and
electrophysiological recordings, by offering regional specificity, high spatial and
temporal resolution and the capability for longitudinal and within subjects design
with trial to trial monitoring of DA release.
Supported by the University of Washington Royalties Research Fund and
National Institutes of Health grants R01-MH079292 (PEMP), R21-DA021793
(PEMP) and F32-DA024540 (JJC).
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