Simultaneous dopamine and single-unit recordings
reveal accumbens GABAergic responses: Implications
for intracranial self-stimulation
Joseph F. Cheer*†, Michael L. A. V. Heien*†, Paul A. Garris‡, Regina M. Carelli§¶, and R. Mark Wightman*¶?
*Department of Chemistry and Neuroscience Center,§Department of Psychology, and¶Curriculum in Neurobiology, University of North Carolina,
Chapel Hill, NC 27599; and‡Department of Biological Sciences, Illinois State University, Normal, IL 61790
Communicated by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, November 4, 2005 (received for review
September 30, 2005)
Intracranial self-stimulation (ICS) is a motivated behavior that
results from contingent activation of the brain reward system. ICS
with stimulating electrodes placed in the medial forebrain bundle
(MFB) is particularly robust. However, the neurons that course
through this pathway use a variety of neurotransmitters including
dopamine and GABA. For this reason, the neurotransmitters that
are central to this behavior, and the specific roles that they
subserve, remain unclear. Here, we used extracellular electrophys-
iology and cyclic voltammetry at the same electrode in awake rats
to simultaneously examine cell firing and dopamine release in the
nucleus accumbens (NAc) during ICS and noncontingent stimula-
tion of the MFB. ICS elicited dopamine release in the NAc and
produced coincident time-locked changes (predominantly inhibi-
tions) in the activity of a subset of NAc neurons. Similar responses
were elicited with noncontingent stimulations. The changes in
firing rate induced by noncontingent stimulations were reversed
by the GABAA receptor antagonist bicuculline. Most time-locked
unit activity was unaffected by D1 or D2-like dopamine-receptor
antagonists, or by inhibition of evoked dopamine release, al-
though, for a minority of units, the D1 dopamine-receptor antag-
onist SCH23390 attenuated neural activity. Thus, neurons in the
NAc are preferentially inhibited by GABAA receptors after MFB
stimulation, a mechanism that may also be important in ICS.
cyclic voltammetry ? electrophysiology ? nucleus accumbens ? motivated
behavior ? behaving rat
related to goal-directed behaviors and contributes to behavioral
selection in response to predictive cues, sequencing of behaviors,
shown that information is rapidly processed by NAc neurons
regardless of reinforcer type (8, 9). A particularly interesting
reinforcer is intracranial self-stimulation (ICS) in which the neural
substrates mediating reward are directly activated (10). In this
paradigm, animals lever press to deliver electrical stimulation to
select brain regions. Like natural rewards, ICS causes time-locked
changes in unit activity in the NAc (11, 12).
The medial forebrain bundle (MFB), a major axonal tract for
both ascending and descending projections, is a site that supports
from their origin in the ventral tegmental area (VTA) (13, 14).
Because electrical stimulation with parameters that are used in ICS
causes antidromic firing of dopaminergic neurons (15) and dopam-
ine-receptor antagonists applied to the NAc inhibit ICS reinforce-
ment (16), dopamine neurotransmission in the NAc is considered
important in reward processing (17). Modern views of reward-
related behavior ascribe an alerting or learning role to dopamine,
rather than direct mediation of hedonia (2, 14, 18, 19). However,
several other neuronal systems project through the MFB and are
activated by its stimulation. For example, a descending, myelinated
he nucleus accumbens (NAc) is an important neural compo-
neuronal pathway has been identified as central to maintenance of
ICS (20). A GABAergic pathway, comprising 36% of VTA pro-
jection neurons (21), ascends to the NAc (22) and can also be
activated in anticipation of a reinforced response for ICS (23).
Because of these complexities, the signaling roles of GABA,
To address this issue, we have combined electrophysiology and
fast-scan cyclic voltammetry at the same carbon-fiber electrode in
awake, unrestrained rats to allow simultaneous monitoring of
neuronal activity and dopamine release at the same location within
the NAc. The combined approach, pioneered by Millar and co-
workers (24), has only been used in anesthetized animals. During
were accompanied by dopamine release. Similar responses were
found with noncontingent delivery of the same stimulation. Be-
(25), pharmacological investigations of the neurotransmitters re-
sponsible for the electrophysiological changes were done with
experimenter-delivered stimulations. The results reveal that the
unit responses are preferentially responsive to GABA, not dopa-
Materials and Methods
Animals and Surgery. Male Sprague–Dawley rats (n ? 28), im-
planted with jugular vein catheters (Charles River Breeding Lab-
oratories) and weighing between 300 and 350 g, were used. Rats,
housed individually, had ad libitum access to food and water with
as described (26). A guide cannula (Bioanalytical Systems, West
Lafayette, IN) was positioned above the NAc core (?1.3 mm
anteriorposterior, ?1.3 mm mediolateral, relative to bregma). An
Ag?AgCl reference electrode was placed in the contralateral hemi-
sphere. A detachable microdrive containing a cylindrical carbon-
fiber microelectrode (50- to 100-?m length of exposed T-650 fiber,
Amoco, Greenville, SC) and the electrode was lowered into the
NAc core. A bipolar stimulating electrode placed above the MFB
(?4.6 mm anteriorposterior, ?1.4 mm mediolateral, and ?7.7 mm
dorsoventral) was lowered in 0.2-mm increments until electrically
evoked dopamine release was detected (between ?8.0 and ?8.8
dental cement. The carbon-fiber electrode was removed and re-
placed with a stylet.
ICS. Experimental sessions began 3 days after surgery in a behav-
ioral chamber (Med Associates, St Albans, VT) that contained a
Conflict of interest statement: No conflicts declared.
Abbreviations: NAc, nucleus accumbens; ICS, intracranial self-stimulation; VTA, ventral
tegemental area; MFB, medial forebrain bundle; PEH, perivent histogram; BIC, bicuculline.
†J.F.C. and M.L.A.V.H. contributed equally to this work.
?To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
December 27, 2005 ?
vol. 102 ?
retractable lever with a cue light above it. For ICS, the animal
a stimulation train to the MFB (24 biphasic pulses, 60 Hz, 125 ?A,
2 ms per phase). The stimulus train was delivered 300 ms after a
lever press. Training began with a 1-s time out (TO1?) in which the
lever was retracted after each press. During the time-out, the cue
light was off, the house light was on, and a 67-dB (1-kHz) tone was
TO10?). Criterion responding was achieved when animals had 30
stimulations per 5-min session, with between one and five sessions
Hz, 125 ?A, 2 ms per phase).
Combined Electrophysiology?Electrochemistry. A carbon-fiber elec-
trode was lowered into the NAc core, and the carbon-fiber and
Ag?AgCl electrodes were connected to head-mounted operational
amplifiers. Signals were routed through an electrical swivel (Med-
Associates), which allowed free movement of the animal, to cus-
tom-built amplifiers (Chemistry Department Electronics Facility,
University of North Carolina, Chapel Hill). Cyclic voltammograms
were generated at 5 Hz by using a triangular waveform (?0.6 to ?
1.4 V, 400 V?s) with custom-written LABVIEW software (National
Instruments, Austin, TX). The electrode was conditioned with the
waveform applied at 60 Hz for 15 min. A solid-state relay in the
headstage alternated between a current amplifier for voltammetric
had a ?20 ms gap every 180 ms when the voltammograms were
collected (Fig. 5, which is published as supporting information on
the PNAS web site). Both signals were referenced to the Ag?AgCl
electrode that was connected to ground. Recordings were made at
sites where single units were isolated and dopamine release could
be detected. After collection of data at one site, the electrode was
lowered ?300 ?m until another unit and release site were found.
Units recorded from carbon-fiber electrodes (impedance at the
tip: ?500 k? at 1 KHz) were amplified (?1,000) and bandpass-
filtered (300–3,000 Hz). They were digitized with commercially
available software (DIGITIZER, Plexon, Dallas). Discrimination of
waveforms was accomplished by using principal component anal-
ysis in OFFLINE SORTER (Plexon). Typically, one to two neurons
could be discriminated at each location. Custom-written software
in the electrochemical record, whereas electrophysiological and
Data Analysis. Dopamine was identified from the cyclic voltammo-
grams that were background subtracted from 10 cyclic voltammo-
grams recorded before the stimulation. They were displayed with
time as the abscissa, voltage as the ordinate, and the background
subtracted current encoded in false color. Dopamine changes were
extracted from the current at the peak for oxidation of dopamine
(approximately ?0.6 V vs. Ag?AgCl) once pH-corrected (27).
Neural activity was characterized with raster displays and
perievent histograms (PEHs) across distinct time domains that
three epochs [baseline (?8 to 0 s before the stimulation), response
(0 to 4 s after stimulation), and recovery (?4 to ?8 s after the
stimulation)] to allow measurement of changes relative to the
stimulation. Statistical differences in unit activity and signal-to-
baseline ratios across epochs were evaluated as described in Sup-
porting Text, which is published as supporting information on the
PNAS web site.
Neuronal Firing Patterns and Dopamine Release Dynamics in the NAc
During ICS. Rats (n ? 11) exhibited stable ICS on the FR1, TO10?
schedule (Fig. 1A Top). Across all animals, the mean latency to
respond after lever extension was 1.1 ? 0.2 s. Simultaneous
electrochemical?electrophysiological measurements were made
during ICS when the animals had reached this level of responding.
voltammograms recorded at one site is shown in Fig. 1A Bottom.
The oxidation of dopamine is apparent by the green features at the
potential where it is oxidized (dashed line). These are seen after
the NAc evoked by ICS (24 pulses, 60 Hz, 125 ?A) of the MFB using an FR1,
TO10? schedule. The top line indicates the time of each reinforced response.
Each lever press caused a stimulation followed by a retraction of the lever for
10 s. The dopamine temporal response, corrected for pH changes, is shown
below. The color representation shows all of the voltammetric currents; the
white dashed line is at the potential for oxidation of dopamine. The green
feature centered on the dashed line is due to the release of dopamine. The
blue features are due to a basic pH shift after dopamine release. A cyclic
voltammogram obtained at the peak of the last response (asterisk) is shown
the ICS lever press (green dashed line). Each tick on the raster plot represents
an extracellularly recorded action potential (average waveform shown to the
top left). The red line is the evoked dopamine signal averaged from the 63
trials shown. Bin width was 200 ms for both measurements.
Responses measured in the NAc during ICS. (A) Dopamine release in
Cheer et al.
December 27, 2005 ?
vol. 102 ?
no. 52 ?
each time out and subsequent lever press. The shape of the
individual cyclic voltammograms further confirms dopamine de-
the color plot. In addition, longer lasting changes (blue features)
accompany the dopamine release that are due to changes in
extracellular pH (27). Removal of the pH component provides the
dopamine concentration evoked by the stimulation (?700 nM in
this example, Fig. 1A). Thus, unlike ICS with a continuous rein-
forcement schedule that resulted in suppression of dopamine
release during the session (28), on the FR1 TO10? schedule, each
lever press and its accompanying stimulus train evoked robust
dopamine release that was stable across the behavioral session.
the electrical stimulation (green dashed line) shown in Fig. 1B
displays the activity of the unit (waveform to the left) that was
simultaneously recorded at the site where the voltammograms
for the 63 trials shown, and the overlaid trace (red) shows the
average of all of the dopamine signals.
Of the 85 NAc neurons recorded during ICS, three categories of
stimulation resulting from the lever press (Fig. 2 Left). U type (n ?
23) were unaffected (Fig. 2, Top Left) with similar firing during the
baseline, response, and recovery epochs (Table 1; F2,68? 0.16; P ?
with an inhibition followed by excitatory components. I?E-type
cells had a mean baseline firing rate that differed significantly from
that measured at the peak but not from that measured in the
recovery epoch (Table 1; F2,59? 2.50; P ? 0.05). I-type (n ? 42),
the most prevalent, were inhibited in the response epoch (Fig. 2
Bottom Left). Onset of recovery from inhibition was 4.7 ? 1.2 s
(mean ? SEM). The amplitude of dopamine release did not differ
Neuronal Firing Patterns and Dopamine Release in the NAc During
Noncontingent MFB Stimulation. Dopamine release and single-unit
activity were also simultaneously monitored in the NAc during
noncontingent (response independent) stimulations (n ? 13 ani-
24 pulses, 60 Hz, 125 ?A). (Right) Neural activity dis-
played in the same way obtained during noncontin-
gent stimulations. Data are shown as composite PEHs.
Histograms were made by summing data from each
unit over individual trials, and sorting according to
responses obtained [Top, unaffected (U-type); Middle,
inhibited?excited (I?E-type); Bottom, inhibited (I-
type)]. The overlaid trace (red) shows the time course
of average extracellular dopamine concentration
changes measured at the same loci where neurons
ms for all histograms.
Table 1. Neuronal firing patterns time-locked to ICS and noncontingent MFB stimulation
24p 60 Hz (23)
1.12 ? 0.3
1.28 ? 0.4
24p 60 Hz (42)
2.48 ? 0.3
1.77 ? 0.2
24p 60 Hz (0)
24p 60 Hz (20)
1.91 ? 0.5
0.62 ? 0.3,
2.77 ? 0.7*
2.12 ? 0.6
24p 60 Hz (12)
0.54 ? 0.2
0.29 ? 0.1,
0.86 ? 0.2*
0.81 ? 0.2
1.05 ? 0.5
24p 60 Hz (24)
1.99 ? 0.8
1.88 ? 0.9
2.32 ? 0.3
24p 60 Hz (46)
0.84 ? 0.1
0.36 ? 0.1
6p 30 Hz (26)
1.26 ? 0.2
1.25 ? 0.5
6p 30 Hz (40)
0.99 ? 0.1
0.28 ? 0.1
6p 30 Hz (14)
2.29 ? 0.5
3.52 ? 0.3
Recovery1.38 ? 0.3 2.04 ? 0.80.57 ? 0.20.52 ? 0.3 2.97 ? 0.5
*Response components are separated into the short inhibition (upper) and the delayed excitation (lower).
www.pnas.org?cgi?doi?10.1073?pnas.0509607102Cheer et al.
mals) using the ICS stimulation train. The composite PEHs are
remarkably similar to those obtained during ICS (Fig. 2 Right). The
majority of NAc cells (n ? 46) were I-type (Fig. 2 Bottom Right,
Table 1; F2,125? 2.19; P ? 0.05), whereas 24 NAc neurons were U
type (Fig. 3, Table 1; F2,71? 0.14; P ? 0.05). The average recovery
time for cell firing of I-type neurons was 5.1 ? 0.6 s. I?E-type (n ?
0. 05). The early excitatory component seen in the composite PEH
Similar activation has been observed during ICS (11, 29). The
maximal dopamine concentration (530 ? 47 nM) did not correlate
recorded (F2,243? 1.6, P ? 0.05).
Pharmacological Effects on NAc Neuronal Inhibitions Time-Locked to
Noncontingent MFB Electrical Stimulation. Dopaminergic effects on
noncontingent, I-type responses were probed by administration of
RO4–1284 (RO; 1 mg?kg?1, i.v.; n ? 7), an inhibitor of vesicular
monoamine transporter 2, the transporter for catecholamine (30),
indoleamine, and histaminergic vesicles. I-type units were selected
because they are the predominant phasic cells and are devoid of
from 243 ? 91 nM to undetectable levels in ?120 s (Fig. 3).
1.87 ? 0.7; t ? ?3.1; P ? 0.05), although baseline firing rate
post-RO: 2.67 ? 0.5 Hz, t ? 10.1; P ? 0.01). This inhibition in
baseline activity was accompanied by a significant decrease in the
signal-to-baseline ratio of the inhibitory response after RO (0.74 ?
0.1 to 0.41 ? 0.2; t ? ?2.6; P ? 0.05).
To investigate GABA responses, after RO administration, bicu-
culline (BIC, a GABAAreceptor antagonist) was administered at
a nonepileptogenic dose (200 ?g?kg?1, i.v.). Surprisingly, this com-
bination further decreased baseline firing rate to 0.54 ? 0.1 Hz (t ?
13.4; P ? 0.0001, Fig. 3C). Presumably, this paradoxical response is
due to the combined presence of BIC and RO that suppresses all
monoamine release. More importantly, I-type responses modified
their patterned discharge to a poststimulation excitation (E-type
response; change from 0.54 ? 0.1 Hz at baseline to 1.17 ? 0.1 Hz
at the peak; t ? ?1.4; P ? 0. 001), whereas evoked dopamine
changes remained undetectable. There was no evidence for anti-
dromic activation because latencies varied from one pulse to the
next (98 ? 16 ms; range 38–125 ms).
Effect of Dopamine Receptor Antagonists on NAc Neuronal Responses
Time-Locked to Short Noncontingent MFB Electrical Stimulation. To
further probe dopamine effects, ‘‘short’’ stimulations (six biphasic
with the long stimulation, t ? 7.5; P ? 0.01), and, therefore, unit
responses mediated by dopamine should be more sensitive to
antagonists. With this stimulation, both U-type (n ? 26) and I-type
as supporting information on the PNAS web site). Again, the firing
rate of U-type cells was not modified by the stimulation (F2,77?
0.07; P ? 0.05). I-type neurons displayed a significant inhibition
relative to stimulation onset (Fig. 4A, Table 1; F2,125? 5.13; P ?
0.05). I?E responses were not observed with the short stimulation
train; however, some neurons (n ? 14) showed E-type responses
(Table 1 and Fig. 6; F2,41? 5.5; P ? 0.05) lasting 3.4 ? 0.4 s. Peak
excitation elicited by I?E cells (Table 1; t ? ?7.1; P ? 0.05).
However, many E-type units (64.2%) were antidromically driven
(11, 29) by the short electrical stimulation as evidenced by their
short and invariant latency to fire (16.7 ? 0.04 ms) after each
stimulus pulse (Fig. 6). Again, the dopamine concentration was
uncorrelated with the different categories of simultaneously re-
corded cells (F2,237? 3.6, P ? 0.05).
Antagonists were administered systemically to further examine
they were given. D1-like dopamine receptor blockade with
SCH23390 (SCH, 40 ?g?kg?1, i.v.; ref. 32) significantly enhanced
the signal-to-baseline ratio for E-type responses (1.24 ? 0.2 to
1.97 ? 0.3; t ? 2.1; P ? 0.05) but not for I-type responses. In
contrast, the D2-like dopamine-receptor antagonist raclopride
(RACLO; 80 ?g?kg?1, i.v.; ref. 32) did not significantly alter
signal-to-baseline ratios (all P values ? 0.05). SCH administration
did not alter I-type (P ? 0.05) but attenuated E-type (P ? 0.05)
firing patterns (Fig. 4A Left). RACLO did not modify the time-
locked response of I-type or E-type (both P ? 0.05) cells (Fig. 4B).
after injection because it significantly elevated evoked maximal
32 nM; t ? ?3.6; P ? 0.01, respectively, Fig. 4 A and B Right),
consistent with its known actions on dopamine release (33).
Here, we adapted technology developed by Millar and coworkers
(24) to obtain the first simultaneous, subsecond measurements of
both dopamine release and neuronal firing in awake, behaving
animals. This approach affords unparalleled insight into both
but modulated by GABAAreceptors. (A) PEH obtained during noncontingent
stimulation (green dashed line at time 0; 24 pulses, 60 Hz, 125 ?A) with
average dopamine response (red line) superimposed. (B) Inhibition of action
potential-driven dopamine release with RO (1 mg?kg?1) decreases back-
ground firing rates, but does not eliminate the inhibition of firing caused by
the stimulation. (C) Blockade of GABAAreceptors with BIC (200 ?g?kg?1) after
RO uncovers a time-locked excitation, whereas background firing is further
decreased and dopamine release remains undetectable (Bottom). Bin width
was 200 ms, all drugs were delivered intravenously.
Inhibitory responses are independent of vesicular dopamine release
Cheer et al.
December 27, 2005 ?
vol. 102 ?
no. 52 ?
chemical and electrical signaling during behavior. Using this tech-
nique, we examined the regulation of NAc neuronal activity and
concurrent dopamine release during ICS, a potent reinforcer.
Although dopamine release is time-locked to the reinforcing elec-
baseline firing. However, the principal finding is that time-locked
inhibitions, the predominant cellular response, are mediated by
Most investigators concur that dopamine neurotransmission in
the NAc plays an essential, but as yet, undefined role in ICS.
Evidence for dopamine’s importance in ICS includes the reduced
responding in rats with lesions of dopaminergic neurons (34), the
blockade of ICS by NAc microinjections of a dopaminergic antag-
onist (16), and the impaired ICS in D1 knockout mice (12).
However, measurements of dopamine release during ICS indicate
that it is not a necessary condition for ICS (35). Indeed, with a
continuous ICS schedule, dopamine release is suppressed (28, 36,
37). Although direct activation of dopaminergic neurons is unnec-
essary for ICS (20), we purposely used stimulation parameters that
eters, used in conjunction with the FR1, TO10? schedule, support
stable dopamine release during ICS and complement those of
Yavich and Tiihonen (37), who showed that stable dopamine
release can occur during ICS on an FR8 schedule.
Robust changes in cell firing of many NAc units were observed
with ICS, noncontingent stimulations with the same waveform,
and short stimulations. In all three cases, the changes in firing
rate were time-locked to the stimulation train and concurrent
with dopamine release. The firing patterns of NAc neurons
during ICS reported here are similar to those reported in mice
during ICS (12, 29). Similarly, dopamine release evoked by ICS
was indistinguishable from the responses observed during non-
contingent stimulations. However, after elimination of dopa-
mine release by using an inhibitor of the vesicular monoamine
transporter (Fig. 3), the inhibitory response was still present.
Similarly, I-type cells were unaltered by D1- or D2-like dopam-
ine-receptor antagonists. The D1-like dopamine-receptor antag-
onist SCH23390 modified baseline firing of E-type cells, a
relatively small subset of all time-locked cells, in a manner
consistent with previous reports (38, 39), and also altered their
time-locked activity. Thus, the results during noncontingent
stimulations suggest a limited role for evoked dopamine on the
observed unit activity.
With noncontingent stimulations similar to the ICS train that
we used, Williams and Millar (40) found that striatal units were
excited upon dopamine release, and the elevated firing contin-
ued for tens of seconds after the stimulation. These responses
were suppressed after dopamine depletion by synthesis inhibi-
tion. Excitation, mediated by D1 receptors, was also observed by
Gonon within 200 ms of the stimulation in a subpopulation of
striatal neurons (41). In contrast to these prior reports in
pamine receptor antagonists on inhibitory and excita-
tory responses time-locked to electrical stimulation of
green dashed line. (A) Time-locked inhibitions of I-
type neurons are unchanged between before (Upper)
?g?kg?1; Left) and raclopride (Raclo, 80 ?g?kg?1,
but Raclo increases electrically evoked release. (B) The
overall response of E-type cells was diminished after
SCH treatment, whereas dopamine release was unal-
tered (Left). Although the time-locked excitation was
unchanged by Raclo treatment (Right), dopamine re-
lease was again increased (Right). Bin width was 200
ms for all PEHs, all drugs were given intravenously.
Composite PEHs showing the effects of do-
www.pnas.org?cgi?doi?10.1073?pnas.0509607102 Cheer et al.