Substantia nigra pars reticulata GABA is involved in the regulation of operant lever pressing: pharmacological and microdialysis studies.

Abstract

Substantia nigra pars reticulata (SNr) is an important mesencephalic nucleus that functions as a relay area for basal ganglia output. SNr receives GABAergic inputs from the neostriatum and globus pallidus, and in turn sends projections to a variety of motor areas. Although a large number of studies have focused upon the behavioral functions of basal ganglia dopamine, much less is known about the behavioral functions of SNr GABA. The present studies were undertaken to investigate the role of SNr GABA in lever pressing behavior. In the first experiment, the GABA(A) antagonist bicuculline was infused locally into SNr to determine if blockade of GABA receptors interfered with the performance of lever pressing on a fixed ratio 5 schedule. SNr injections of bicuculline produced a dose-related suppression of operant responding. Analysis of interresponse time bins showed that SNr bicuculline produced a response slowing characterized by a relative reduction in the number of fast interresponse times, and an increase in the relative number of pauses. In an additional experiment, microdialysis methods were used to determine if extracellular GABA is elevated during the performance of fixed ratio five lever pressing. During the 30 min lever pressing session, extracellular GABA showed a significant and substantial increase relative to baseline levels. These data support the hypothesis that SNr GABA is involved in the regulation of motor output, and indicate that GABA release in this structure is increased during behavioral stimulation.

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SUBSTANTIA NIGRA PARS RETICULATA GABA IS INVOLVED IN THE
REGULATION OF OPERANT LEVER PRESSING: PHARMACOLOGICAL
AND MICRODIALYSIS STUDIES
M.
A. WISNIECKIaAND J. D. SALAMONEa*
CORREA,a,b
S.MINGOTE,a
A.BETZ,a
aDepartment of Psychology, University of Connecticut, Storrs, CT,
06269-1020, USA
bArea de Psicobiologia, Campus Riu Sec, Universitat Jaume I, 12071,
Castello, Spain
Abstract—Substantia nigra pars reticulata (SNr) is an impor-
tant mesencephalic nucleus that functions as a relay area for
basal ganglia output. SNr receives GABAergic inputs from
the neostriatum and globus pallidus, and in turn sends pro-
jections to a variety of motor areas. Although a large number
of studies have focused upon the behavioral functions of
basal ganglia dopamine, much less is known about the be-
havioral functions of SNr GABA. The present studies were
undertaken to investigate the role of SNr GABA in lever
pressing behavior. In the first experiment, the GABAAantag-
onist bicuculline was infused locally into SNr to determine if
blockade of GABA receptors interfered with the performance
of lever pressing on a fixed ratio 5 schedule. SNr injections of
bicuculline produced a dose-related suppression of operant
responding. Analysis of interresponse time bins showed that
SNr bicuculline produced a response slowing characterized
by a relative reduction in the number of fast interresponse
times, and an increase in the relative number of pauses. In an
additional experiment, microdialysis methods were used to
determine if extracellular GABA is elevated during the per-
formance of fixed ratio five lever pressing. During the 30 min
lever pressing session, extracellular GABA showed a signif-
icant and substantial increase relative to baseline levels.
These data support the hypothesis that SNr GABA is in-
volved in the regulation of motor output, and indicate that
GABA release in this structure is increased during behavioral
stimulation. © 2003 IBRO. Published by Elsevier Science Ltd.
All rights reserved.
Key words: basal ganglia, motor, Parkinson’s disease,
dialysis, bicuculline, GABAA.
Substantia nigra pars reticulata (SNr) is a mesencephalic
nucleus that functions as a relay area for basal ganglia
output (Bevan et al., 1996; Bolam et al., 2000). Although
pars compacta contains dopamine (DA) cell bodies that
project to neostriatum, the SNr is ventral to the compacta,
and receives GABAergic projections from neostriatum and
lateral globus pallidus (Parent et al., 1984; Loopjuit and
Van der Kooy, 1985; Gerfen, 1992; Parent and Hazrati,
1993; Fallon and Laughlin, 1995). In turn, GABAergic neu-
rons that originate in SNr project to thalamic motor nuclei,
superior colliculus and brainstem motor areas (Faull and
Mehler, 1978; Fallon and Laughlin, 1995; Kha et al., 2001).
Although a large number of studies have focused upon the
behavioral functions of forebrain DA systems, much less is
known about the behavioral functions of SNr GABA. Phar-
macological evidence indicates that GABA in SNr is in-
volved in various aspects of motor function (Di Chiara et
al., 1978; Scheel-Kruger et al., 1981a, 1981b; Scheel-
Kruger, 1983; Cools et al., 1983; Baumeister et al., 1988;
Finn et al., 1997; Kriem et al., 1998; Mayorga et al., 1999;
Koch et al., 2000; Wichmann et al., 2001). Transplantation
of engineered GABA-releasing cells into SNr was shown to
reduce tremulous movements induced by cholinergic stim-
ulation (Carlson et al., in press). Recent studies in rats
indicated that local infusions of the GABAAagonist musci-
mol into SNr increased locomotion and circling behavior,
and that injections of the GABAAantagonist bicuculline
decreased open field locomotion and motor activity in
small test chambers (Trevitt et al., 2002).
The present studies were undertaken to investigate the
role of SNr GABA in lever pressing behavior. Lever press-
ing was studied because this behavior is known to be
sensitive to antagonism or depletion of DA in the basal
ganglia (Nowend et al., 2001; Salamone et al., 1993, 1997,
1999, 2001; Trevitt et al., 2001), and also because lever
pressing is associated with increases in extracellular DA
activity in striatum and nucleus accumbens (Cousins and
Salamone, 1996; Salamone et al., 1999; McCullough et al.,
1993; Salamone et al., 1989, 1994; Sokolowski et al.,
1998; see reviews by Salamone, 1996; Salamone et al.,
1999). Two experiments were performed in the present
investigation. In the first experiment, the GABAAantago-
nist bicuculline was infused locally into SNr to determine if
blockade of GABA receptors would interfere with the per-
formance of lever pressing on a fixed ratio 5 schedule. A
GABAAantagonist was used in this experiment because
there are a greater number of GABAAreceptors than
GABABreceptors on cell bodies in the SNr (Bowery et al.,
1987; Nicholson et al., 1992; Ng and Yung, 2000), and
because most of the GABABreceptors in SNr are thought
to be located on either presynaptic terminals as autorecep-
tors, or on the dendrites of SNc DA neurons (Giralt et al.,
1990; Nicholson et al., 1992; Shen and Johnson, 1997; Ng
and Yung, 2000). In addition, previous studies have shown
that drugs acting on GABAAreceptors have very potent
behavioral effects when injected into SNr (Di Chiara et al.,
*Corresponding author. Tel: ?1-860-486-4302; fax: ?1-860-486-
2760.
E-mail address: salamone@psych.psy.uconn.edu (J. D. Salamone).
Abbreviations: SNr, substantia nigra pars reticulata; DA, dopamine;
FR5, fixed ratio 5; IRT, interresponse time; OPA, o-phthalidialdehyde;
HPLC, high-performance liquid chromatography; aCSF, artificial cere-
brospinal fluid; ANOVA, analysis of variance; TTX, tetrodotoxin.
Neuroscience 119 (2003) 759–766
0306-4522/03$30.00?0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0306-4522(03)00117-9
759

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1978; Scheel-Kruger et al., 1981a, 1981b; Scheel-Kruger,
1983; Starr and Summerhayes, 1983; Baumeister et al.,
1988; Amalric and Koob, 1989; Starr and Starr, 1989, Finn
et al., 1997; Kriem et al., 1998; Mayorga et al., 1999). In
the present experiment, the computer program that con-
trolled the lever pressing sessions also recorded the dis-
tribution of interresponse times in order to characterize the
temporal patterns of responding. The interresponse time
(IRT) is the reciprocal of the local rate of responding, and
this measure has been used in several previous studies to
provide a detailed characterization of the effects of striatal
or accumbens DA depletions on lever pressing (Salamone
et al., 1993, 1995, 1999; Sokolowski and Salamone,
1998). Because previous studies have shown that block-
ade of GABAAreceptors in SNr results in decreased motor
activity (Trevitt et al., 2002), it was hypothesized that bicu-
culline injected into SNr should decrease lever pressing
and lead to a slowing of the IRT distribution. In the second
experiment, microdialysis methods were used to deter-
mine if extracellular GABA is elevated during the perfor-
mance of fixed ratio five lever pressing. In view of the
previous work indicating that stimulation of GABAArecep-
tors in SNr is associated with antiparkinsonian effects
(Wichmann et al., 2001) and increases in motor activity
(Trevitt et al., 2002), it was hypothesized that the perfor-
mance of lever pressing behavior would be accompanied
by increases in extracellular GABA in SNr.
EXPERIMENTAL PROCEDURES
Subjects
Male Sprague–Dawley rats (Harlan Sprague Dawley, Indianapo-
lis, Ind.) weighing 280–330 g were used (total n?58). The rat
colony was maintained at 23 °C with a 12-h/12-h light/dark cycle
(lights on 0700 h). Before surgery, rats were group housed; after
surgery, rats were housed separately. All efforts were made to
minimize the number of animals used, and their suffering. Animal
protocols were approved by the institutional animal care commit-
tee, and the methods were in accordance with the Guide for the
Care and Use of Laboratory Animals, Institute of Laboratory Ani-
mal Resources, Commission on Life Sciences, National Research
Council, National Academy Press (1996).
Drugs
Bicuculline methbromide was purchased from Research Bio-
chemicals International (RBI, Natick, MA). Xylazine and Ketamine
were purchased from Phoenix Pharmaceutical, Inc. (St. Joseph,
MO). Acepromazine maleate was purchased from Boehringer
Ingelheim Vetmedica, Inc. (St. Joseph, MO).
Surgical procedures
For cannulae implantations in both the drug and the dialysis experi-
ments, rats were anesthetized with a solution (1.0 mL/kg, i.p.) that
contained Ketamine (100 mg/ml) and Xylazine (20 mg/ml). The inci-
sor bar on the stereotax was set to 5.0 mm above the interaural line.
For the intracranial drug injection study, rats were implanted with
bilateral 23 ga stainless steel guide cannulae. For implantation of
dialysis guide cannulae, rats received unilateral implantation (with
side balanced across rats) of a 10-mm guide cannula purchased
from Bioanalytical Systems, Inc. (BAS; West Lafayette, Ind.). All
guide cannula placements for drug infusion and dialysis were im-
planted 2.0 mm above the rostral SNr at the following coordinates
(Pellegrino and Cushman, 1967): AP ?3.0 mm (from bregma),
LM?1.8 mm (from midline), DV ?7.2 mm (from skull surface). For all
types of implantations, the guide cannulae were secured to the skull
using stainless-steel screws and cranioplastic cement. A stainless-
steel stylet was inserted through each cannula to ensure its patency
throughout the experiment.
Dialysis probe insertion and sampling procedure
After implantation of guide cannulae, rats were singly housed and
were allowed to recover 7 days before testing was resumed. Next,
rats were retrained on the FR5 schedule for approximately two
weeks, and all rats reached a stable baseline as described above.
Prior to insertion of the dialysis probe, rats were given an injection
of 1.5 mg/kg acepromazine maleate to produce mild sedation.
Each rat then had a concentric dialysis probe (2.0 mm active
dialysis surface at tip; also purchased from BAS) inserted through
the guide cannula. After insertion, the probe extended 2.0 mm
beyond the guide cannula, with the active surface located in the
SNr. At the time of implantation, the dialysis probe was attached
via polyethylene tubing to a fluid swivel. Artificial cerebrospinal
fluid (aCSF) was continuously perfused through the tubing at a
rate of 2.0 ?L/min.
Neurochemical analysis of GABA
GABA was assayed using high-performance liquid chromatogra-
phy (HPLC) and electrochemical detection (ESA, New Bedford,
MA). The HPLC system comprised a Waters dual-piston pump, a
precolumn filter, a reverse-phase column (Velosep, C-18,
3x100 mm), an electrochemical detector (Coulochem model
5014A), and a data acquisition station (ESA). The HPLC analyses
used were developed in our laboratory (Trevitt et al., 1999, 2002),
and were modified from those of Phillips and Cox (1997). Electro-
chemical parameters for GABA detection were as follows: detec-
tor 1??0.35 V, detector 2??0.60 V, guard cell??0.65 V. Pre-
injection derivitization of GABA was accomplished using o-phtha-
lidialdehyde (OPA) with sulfite as the nucleophile. GABA, OPA,
and sulfite react to form a stable, electrochemically active GABA-
isoindole sulfonate derivative. Fifty microliters of dialysate or a
standard solution (10?7M GABA) and 20 ?L derivatizing reagent
(22.0 mg OPA dissolved in 0.5 mL ethanol, with 0.5 mL 0.0313 M
sodium sulfite and 9.0 mL 0.1 M sodium tetraborate added) were
mixed and allowed to react for 15 min. The sample was then
assayed using HPLC [mobile phase?100 mM sodium phosphate
buffer, 8% methanol, 10.0 ?L 4.0 M ethylene diamine tetraacetic
acid, set to pH 4.9; flow rate?1.0 mL/min]. Twenty-five minutes
after injection of the reacted sample, an injection of 40 ?L ethanol
was made to remove late-eluting peaks. Extensive work from our
laboratory has shown that these derivatization and chromato-
graphic methods allow for the reliable separation of GABA from
numerous other peaks in dialysates obtained from SNr samples
(Trevitt et al., 1999, 2002). Standards of GABA were assayed
before and after the dialysis samples.
Effect of bicuculline on lever pressing
Male Sprague–Dawley rats initially restricted to 85% of their free-
feeding weight were trained to lever press on a continuous sched-
ule (one 45 mg pellet per lever press). After 1 week, rats were
switched to a fixed ratio 5 schedule (FR5; one pellet per five lever
presses) and tested in 30-min sessions, 5 days/week for 3 weeks.
After this initial training, the rats underwent bilateral implantation
of cannulae. After recovery from surgery, rats were retrained on
the FR5 schedule for an addition few weeks (usually 2–3 weeks),
until FR5 responding was relatively stable (i.e. less than 25%
variability for at least three days), and was maintained at relatively
high levels (i.e. ?1200 responses per 30 min). On operant test
days and on days off, rats received supplemental feeding in their
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home cages to maintain their body weight, with the amount vary-
ing depending upon the weight of the rat and the amount of lever
pressing on that day. After post-surgical testing was complete,
rats were randomly assigned to one of four groups. Rats received
injections of either vehicle (n?10) or bicuculline (4.5, 9.0 or 18.0
ng/0.5 ?L per side; n?8, 10, 8 respectively). Bicuculline was
dissolved in 0.9% saline solution, which also served as the vehicle
control. Vehicle or drug infusions were delivered by a 30-ga injec-
tor that extended 2.0 mm beyond the cannula tip. Each injector
was attached to a 10.0 ?L Hamilton syringe via PE 10 tubing, with
the injections driven by a Harvard syringe pump. All injections
were at a volume of 0.5 ?L per side, at a flow rate of 0.5 ?L/min,
and the injector was left in place for 1 min after injection. Each
animal received an injection of only one drug treatment condition.
Directly following injection procedure, the animals were placed in
the operant chamber for a 30-min FR5 session. A BASIC program
designed to run this schedule recorded the total number of lever
presses, and also recorded each interresponse time (IRT; i.e. the
time between onset of one response and onset of the next one),
and placed each IRT within a time bin (0–0.5 sec, 0.5–1.0 sec,
etc., up to 4.5–5.0 sec, and a final time bin of ?5.0 sec).
Microdialysis study
Rats were trained as described above, and then a single unilateral
guide cannula was surgically implanted above the SNr. After
recovery from this surgery there was a post-surgical training pe-
riod, during which the rats were exposed to simulated dialysis runs
(i.e. a baseline with no lever in the chamber, followed by a 30-min
FR5 session during which the lever was inserted into the chamber
and the lights were dimmed, followed by a brightening of the room
and removal of the lever). After this final stage of training, rats had
dialysis probes inserted (BAS) through the guide cannulae. Neu-
rochemical samples were collected every 30 min on the day
following probe implantation. Artificial cerebrospinal fluid (aCSF;
147.2 mM NaCl, 2.4 mM CaCl2, 4.0 mM KCl) was pumped
through the probe at a rate of 2.0 ?L/min. Four baseline sessions
were conducted prior to the behavioral test, and then a 30-min
FR5 lever pressing session was performed during which a single
sample was collected. During the baseline period there was no
lever protruding into the chamber. The lever was inserted into the
chamber, and the lights were dimmed, to signify the onset of the
behavioral session to the animal, and then the lights were turned
on and the lever removed at the end of the lever pressing session.
Additional samples were collected after the behavioral session.
Samples were frozen and later analyzed for GABA content using
reverse-phase HPLC with electrochemical detection as described
above. After the last sample, the probes were removed and the
rats were anesthetized and perfused.
Histology
Several days after the final testing session, each animal was
anesthetized with CO2and perfused with physiological saline
followed by a 3.7% formaldehyde solution. The brains were re-
moved and stored in formaldehyde, and then were cut in 50-?m
slices and mounted on glass microscope slides. Following slicing,
slides were stained with Cresyl Violet, and cannulae placements
were determined using a microscope.
Data analysis
For the bicuculline study, data on total numbers of responses were
analyzed with between-groups analysis of variance (ANOVA), and
non-orthogonal planned comparisons were conducted in which each
dose group was compared against the vehicle control group. IRT bin
data were analyzed by 4?11 factorial ANOVA, with repeated mea-
sures on the bin factor. For these analyses, the data were calculated
as percent of IRTs in each bin, in order to correct for drug-induced
changes in the number of responses, and to allow for analysis of the
relative frequency distribution of IRTs. Because ANOVA revealed
that there was a significant dose x bin interaction, each bin was
analyzed separately by analysis of simple effects (Keppel, 1982),
and the error terms used were restricted to the specific groups being
compared because of heterogeneity of variance. Within the two bins
that showed significant dose effects, planned comparisons were
conducted between each drug treatment group and the vehicle
group. For the microdialysis study, picogram levels of GABA in the
dialysis samples were analyzed by repeated measures ANOVA,
which included the last three baseline samples, the sample taken
during the behavioral session, and the three samples taken after the
behavioral session. After the determination of the significance of the
overall ANOVA, non-orthogonal planned comparisons (Keppel,
1982) were used to compare each sample with the last baseline
sample, which was used as the control.
RESULTS
Injections of bicuculline into SNr resulted in dose-related
suppression of lever pressing (Fig. 1). ANOVA revealed that
there was a significant effect of bicuculline treatment on lever
pressing (F(3,32)?10.8, P?0.001), with the 9.0 and 18.0 ng
doses being significantly suppressed relative to vehicle. An
examination of the number of lever presses across the three
10-min intervals in the session indicates that control animals
continue to respond throughout the session (1st10 min,
mean?439;2nd
10min,
mean?551). Bicuculline did not produce a within-session
decline in overall responding, and although there was not
a significant dose x time interaction (data not shown), the
18 ng dose tended to have its greatest effects in the first 10
min (i.e. responding suppressed by 18 ng bicuculline to
24.6% of control) as opposed to the last 10 min (respond-
ing suppressed to 54.1% of control), which suggests that
the drug effect is relatively short-acting. Detailed analyses
of the relative distribution of interresponse times (Fig. 2)
demonstrated that SNr bicuculline produced substantial
alterations in the pattern of IRTs shown. Factorial ANOVA
mean?553;3rd
10min,
Fig. 1. Dose response curve for the effect of SNr injections of bicu-
culline on total number of lever presses (mean?S.E.M. number of
lever presses shown). Rats received injections of either vehicle (n?10)
or bicuculline (4.5, 9.0 or 18.0 ng/0.5 ?L per side; n?8, 10, 8 respec-
tively). Bicuculline-treated rats showed significant reductions in lever
pressing compared with control levels (* different from control,
P?0.05).
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revealed that there was a significant dose x IRT bin inter-
action (F(30,320)?4.0, P?0.001). Bicuculline produced a
dose-related decrease in the relative number of IRTs in bin
one (0.0–0.5 sec; F(3,32)?4.42, P?0.01), which indicates
a decrease in the fastest type of responding (i.e. IRTs
0–250 ms). Within this bin, the 18.0 ng group differed
significantly from vehicle (P?0.05). In addition, there was a
significant increase in the relative number of IRTs in the
last time bin (?5.0 sec; F(3,32)?7.56, P?0.01), which
indicates a relative increase in the number of pauses in
responding. The 18.0 ng group differed significantly from
vehicle within this bin as well (P?0.05). Fig. 3 shows the
results from the analysis of histology to determine place-
ment location in the animals that received 9.0 and 18.0 ng
bicuculline. At the 18.0 ng dose of bicuculline, five animals
were rejected because they had placements that were
either toomedial,too
(mean?S.E.M.) responses?1150.0 (?222.5). Overall, 16
animals (30.8% of the total operated) were rejected be-
cause of bad placements, asymmetry, or excessive dam-
age at the injection site. All n values listed above, and all df
and ANOVA values, were based solely upon animals that
had verified placements in SNr.
In the dialysis experiment, there was a substantial and
significant increase in extracellular GABA during FR5 lever
pressing in the six rats that had histologically verified
placements in the SNr (Fig. 4). During the 30-min test
session, rats with probe placements in the SNr showed
substantial numbers of lever presses (mean lever press
responses?1264 per 30 min). ANOVA with repeated mea-
sures demonstrated that there was a significant overall
differenceacross samples
Planned comparisons demonstrated that the sample col-
lected during the behavioral session significantly differed
from the last baseline sample (F(1,30)?12.8, P?0.01). In
dorsal,or tooanterior
(F(6,30)?3.4,
P?0.05).
addition, the sample collected after the behavioral session
also significantly differed from the last baseline sample
(F(1,30)?5.7.8, P?0.05).
DISCUSSION
The results of the first experiment demonstrated that bilateral
injections of bicuculline into SNr produced a dose-related
suppression of lever pressing. Bicuculline was very potent at
producing this effect, with 18.0 ng producing a suppression of
responding that was greater than 60%. Analysis of the distri-
bution of IRTs demonstrated that bicuculline-induced sup-
pression of responding was characterized by a dose-related
reduction in the relative number of fast IRTs (i.e. 0.0–0.5
sec). In addition, bicuculline increased the relative number of
slow IRTs (i.e. ? 5.0 sec), which typically reflect pauses in
lever pressing. Taken together, this pattern of results sug-
gests that SNr GABAAantagonism produces a shift in the
relative frequency distribution of IRTs that is consistent with
an overall slowing and fragmentation of responding. It should
be pointed out that this pattern of alteration of the IRT distri-
bution is not characteristic of every manipulation that reduces
leverpressing.Forexample,extinction(withdrawalofreward)
tends to increase the relative number of fast IRTs, which is
thought to reflect the extinction “burst” (Salamone et al.,
1995). In addition, pre-feeding to reduce food motivation was
shown to greatly suppress responding on a FR64 schedule,
yet this manipulation had no effect on the overall IRT distri-
bution (Salamone et al., 1999). Nevertheless, the pattern of
effects shown by bicuculline in the present experiment (i.e.
reduction in the relative number of fast IRTs coupled with
increases in pauses) has been shown to result from systemic
DA antagonism (Salamone et al., 1993), and from depletions
of DA in nucleus accumbens (Salamone et al., 1993, 1995,
1999; Sokolowski and Salamone, 1998) and ventrolateral
Fig. 2. Mean?S.E.M. the relative number of interresponse times (IRTs; expressed as percent of total IRTs) within each 0.5 sec time bin up to 5.0 sec,
as well as the percentage of IRTs that were greater than 5.0 sec (* different from control in that time bin, P?0.05).
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neostriatum (Salamone et al., 1993; Cousins and Salamone,
1996). Thus, antagonism of GABAAreceptors in SNr, like
blockade DA receptors and depletions of accumbens and
striatal DA, suppresses total response output and slows the
temporal pattern of responding. This pattern of effects is
consistent with the involvement of basal ganglia circuitry,
including GABAergic transmission in SNr, in the regulation of
aspects of motor function that are necessary for producing
high rates of lever pressing. Future research should employ
schedules that generate low baseline rates of responding
(e.g. fixed interval 1 or 2 min) to determine if infusions of the
GABAAagonist muscimol into SNr could act to increase
response rates.
The results of the present experiment with bicuculline
are consistent with previous research showing that local
SNr injections of drugs that act on GABAAreceptors can
exert potent effects on motor output (Scheel-Kruger et al.,
1981a,b; Baumeister et al., 1988; Amalric and Koob, 1989;
Timmerman and Abercrombie, 1996; Finn et al., 1997;
Kriem et al. 1998; Mayorga et al., 1999; Wichmann et al.,
2001). Several previous studies from our laboratory have
demonstrated that bicuculline can have substantial effects
on aspects of motor function when injected into SNr in the
same dose range used in the present paper. Some studies
have focused upon GABAergic modulation of cholinomi-
metic-induced jaw movements, which have been sug-
gested as a rodent model of parkinsonian tremor (Sala-
mone et al., 1998). For example, 6.0 ng of bicuculline
injected into SNr led to an induction of tremulous jaw
movements (Salamone et al., 1998). Bicuculline infused
into SNr at doses of 6.0–12.0 ng was shown to reverse the
D1-agonist induced suppression of cholinomimetic-stimu-
lated tremulous movements (Mayorga et al., 1999). In
addition, doses of 12.0–18.0 ng bicuculline injected into
SNr were shown to suppress locomotor activity in the open
field, and in small stabilimeter cages (Trevitt et al., 2002).
Thus, together with the present data, these results suggest
that blockade of GABAAreceptors in SNr can lead to motor
effects that are indicative of various features of Parkinson-
ism (i.e. motor slowing, reduced locomotion, tremulous
movements). Although the specific cellular circuitry that
mediates the effects of SNr injections of bicuculline is not
known (see Tepper et al., 1995; Cobb and Abercrombie,
2002), the nature of the effect (i.e. suppression of motor
activity) suggests that these actions are not related to
increased dopamine release in striatum. The present find-
ings are consistent with current models of basal ganglia
function suggesting that hyperactivity of projection neurons in
output structures such as medial globus pallidus and SNr is
related to the production of parkinsonian symptoms (Delong,
Fig. 3. Schematic drawing showing placements of injectors for the
rats that received 9.0 ng (open cylinders) and 18.0 ng (black trape-
zoids) bicuculline in SNr. One side of the brain is shown.
Fig. 4. Mean?S.E.M. levels of GABA in successive 30 min dialysis
samples obtained during the baseline period (BL1–BL3), during the
30-min FR5 lever pressing session (FR5), and during the period after
the behavioral session (post1–post3). (** different from last baseline,
P?0.01; * different from last baseline, P?0.05).
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Page 6

1990; Vila et al., 1996; Abercrombie and De Boer, 1997;
Hauber, 1998; Wichmann et al., 1999, Obeso et al., 2000).
The microdialysis experiment demonstrated that per-
formance of the FR5 schedule was accompanied by in-
creases in extracellular GABA in SNr. The increase in
extracellular GABA during the lever pressing session, rel-
ative to the last baseline sample, was approximately 40%.
Extracellular levels of GABA remained somewhat elevated
during the sample collected after the lever pressing ses-
sion, and returned to baseline levels during the last two
samples. The overall magnitude and duration of the in-
crease in extracellular GABA that was observed in the
present experiment was comparable to the types of
changes in extracellular DA that have been observed in
nucleus accumbens and neostriatum during the same be-
havioral procedure (Salamone et al., 1994; Cousins and
Salamone, 1996; Sokolowski et al., 1998). It is possible
that the elevation of extracellular GABA in SNr during lever
pressing reflects an increase in the neuronal activity of
GABAergic inputs into SNr, such as striatonigral and pal-
lidonigral neurons. If this were true, then it would mean that
GABAergic tone in SNr is not merely gating motor function
in a permissive manner, but instead is actively participating
in the regulation of motor output by undergoing fluctuations
in dynamic activity. This hypothesis is consistent with sev-
eral electrophysiology studies demonstrating that many
striatal and pallidal neurons show increased activity during
the performance of instrumental behaviors such as lever
pressing (Patino et al., 1995; Carelli et al., 1997; Holler-
man, et al., 2000; Ono et al., 2000). Although the neurons
recorded in these studies were not specifically identified as
projecting to SNr, it is nevertheless true that much of the
GABA in SNr comes from projection neurons originating in
these areas. In addition, the present results are consistent
with previous studies showing that DA release is increased
in striatal areas during lever pressing (McCullough et al.,
1993; Salamone et al., 1989, 1994; Cousins and Sala-
mone, 1996, 1999; Sokolowski et al., 1998; see reviews by
Salamone et al., 1994; Salamone et al., 1999), and that
stimulation of DA D1 receptors or glutamate receptors in
striatum can activate striatal neurons (Gonon, 1997) and
increase GABA release in SNr (Biggs et al., 1995; Morari
et al., 1996; O’Connor, 1998; Bianchi et al., 1998).
In addition to being sensitive to behavioral conditions
such as FR5 lever pressing, recent studies from our labo-
ratory indicate that extracellular levels of GABA can be
altered by behaviorally-relevant pharmacological manipu-
lations as well. For example, direct infusions of the D1
agonist SKF 82958 into SNr were shown to increase local
extracellular GABA levels (Trevitt et al., 2002). A behav-
iorally active dose of the GABA uptake inhibitor ?-alanine
significantly elevated extracellular GABA in SNr (Ishiwari
et al., 2002). Nevertheless, in evaluating these result, as
well as the present work, one also should realize the
limitations of this type of dialysis study on SNr GABA.
Indeed, there is considerable controversy about the pos-
sible sources of extracellular GABA that are measured in
dialysis experiments (Timmerman and Westerink, 1997).
There is evidence indicating that basal levels of extracel-
lular GABA are not tetrodotoxin (TTX)-dependent (Biggs et
al., 1995; Timmerman and Westerink, 1995, 1997), while
other studies have shown that GABA levels can be de-
creased slightly by TTX infusion (See and Berglind, 2001).
Recent research from our laboratory, employing the same
methods as those used in the present study, indicated that
local infusions of 1.0 ?M TTX through the dialysis probes
resulted in a statistically significant but very small decrease
in extracellular GABA in SNr (i.e. 15% decrease from
baseline; Mingote, Salamone, et al., unpublished observa-
tions). Thus, unlike other transmitters such as DA in nu-
cleus accumbens and neostriatum (e.g. Cousins et al.,
1999), extracellular GABA levels in SNr are either insen-
sitive, or are only minimally sensitive, to TTX. Interpreta-
tions of the results of TTX infusions during behavioral
performance also would involve considerable difficulties,
because TTX infusions into SNr can produce motor impair-
ments that disrupt the behavior being studied. Thus, the
precise source of the increase in extracellular GABA in the
present study is unknown, and it is possible that increases
in extracellular GABA occur in SNr because of dendritic
release of DA onto GABA terminals (Trevitt et al., 2001,
2002), because of local modulation of GABA uptake, or
also because of GABA released from non-neuronal
sources, such as glia. Yet despite any debate about the
physiological significance of the various sources of extra-
cellular GABA and the uncertainties about the specific
mechanisms involved, the present results do suggest that
behavioral activity can be associated with increases in
extracellular GABA in SNr, at least under some conditions.
This observation, coupled with the pharmacological data
showing that bicuculline is highly potent in suppressing
lever pressing, support the hypothesis that SNr GABA
mechanisms are involved in the modulation of lever press-
ing behavior and other motor activities.
CONCLUSIONS
The results of the first experiment demonstrated that injec-
tions of very low doses of the GABAAantagonist bicuculline
into SNr substantially suppressed lever pressing on a FR5
schedule. Analyses of the IRT distributions in vehicle and
bicuculline-treated rats indicated that bicuculline produced a
general slowing and fragmentation of the temporal pattern of
responding. In addition, the microdialysis experiment showed
that responding on the FR5 schedule is accompanied by
substantial increases in extracelluar GABA in SNr. These
data support the hypothesis that SNr GABA is involved in the
regulation of motor functions that are involved in lever press-
ing, and indicate that GABA release in this structure is in-
creased during behavioral stimulation. It is possible that the
increased GABA release during lever pressing could contrib-
ute to the disinhibitory processes proposed in several models
of basal ganglia function (e.g. Delong, 1990; Hauber, 1998).
Alternatively, it is possible that increased SNr GABA release
during lever pressing acts as a filter, or a modulator of signal-
to-noise ratio, by inhibiting some neurons, while at the same
time altering the selectivity or sensitivity of the SNr neurons
that are activated during operant behavior (Gulley et al.,
M. Correa et al. / Neuroscience 119 (2003) 759–766 764

Page 7

2002; Ferre et al., 1996; Young and Penney, 1993; Wich-
mann and DeLong, 1996; Trevitt et al., 1994; Matuszewich
and Yamamoto, 1999; King 1999).
Acknowledgements—This work was supported by grants to J.S.
from NIH/NINDS, to M.C. from Generalitat Valenciana, Post 00-
09-137, Spain, and to S.M. from the Fulbright Foundation.
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(Accepted 21 January 2003)
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