Dopamine inhibits GABA transmission from the globus pallidus to the thalamic reticular nucleus via presynaptic D4 receptors.

Abstract

The globus pallidus sends a significant GABAergic projection to the thalamic reticular nucleus. Because pallidal neurons express D4-dopamine receptors, we have explored their presence on pallidoreticular terminals by studying the effect of dopamine and D4-receptor agonists on the GABAergic transmission in the thalamic reticular nucleus. We made whole-cell recordings of inhibitory postsynaptic currents (IPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in the thalamic reticular neurons. Dopamine consistently reduced the IPSCs. The effect of dopamine was associated with paired-pulse facilitation, indicating a presynaptic location of the receptors. The effect of dopamine was also measured on the mIPSCs, reducing their frequency but not affecting their amplitude, which also suggests a presynaptic site of action. The selective D4-receptor agonist PD 168,077 also reduced the IPSCs, which was also associated with paired-pulse facilitation. In addition, this agonist reduced the frequency of the mIPSCs with no effect on their amplitude. The D4-receptor antagonist L-745,870 totally blocked the effect of the D4-receptor agonist, indicating the specificity of its effect. To verify the location of the receptors on the pallidal terminals, these were eliminated by injecting kainic acid into the globus pallidus. Kainic acid produced a drastic (80%) fall in the globus pallidus neuronal population. In this condition, the effect of the activation of D4 receptors both on the IPSCs and mIPSCs was prevented, thus indicating that the location of the receptors was on the pallidal terminals. Our results demonstrate that dopamine controls the activity of the thalamic reticular neurons by regulating the inhibitory input from the globus pallidus.

Full text

Page 1

DOPAMINE INHIBITS GABA TRANSMISSION FROM THE GLOBUS
PALLIDUS TO THE THALAMIC RETICULAR NUCLEUS VIA
PRESYNAPTIC D4 RECEPTORS
D. GASCA-MARTINEZ,aA. HERNANDEZ,bA. SIERRA,c
R. VALDIOSERA,cV. ANAYA-MARTINEZ,dB. FLORAN,c
D. ERLIJcAND J. ACEVESc*
aDepartamento de Farmacología, CINVESTAV-IPN, Apartado postal
14-740, México D.F., 07000, México
bDivision de Neurociencias, Instituto de Fisiología Celular UNAM,
Apartado postal 70-253, México D.F., 04510, México
cDepartamento de Fisiología, Biofísica y Neurociencias, CINVESTAV-
IPN, Apartado postal 14-740, México D.F., 07000, México
dDepartamento de Neurociencias, UNAM-Iztacala, Apartado postal
314, Los Reyes Iztacala, Edo. de México, 54090 México
Abstract—The globus pallidus sends a significant GABAergic
projection to the thalamic reticular nucleus. Because pal-
lidal neurons express D4-dopamine receptors, we have ex-
plored their presence on pallidoreticular terminals by study-
ing the effect of dopamine and D4-receptor agonists on the
GABAergic transmission in the thalamic reticular nucleus.
We made whole-cell recordings of inhibitory postsynaptic
currents (IPSCs) and miniature inhibitory postsynaptic cur-
rents (mIPSCs) in the thalamic reticular neurons. Dopamine
consistently reduced the IPSCs. The effect of dopamine was
associated with paired-pulse facilitation, indicating a presyn-
aptic location of the receptors. The effect of dopamine was
also measured on the mIPSCs, reducing their frequency but
not affecting their amplitude, which also suggests a presyn-
aptic site of action. The selective D4-receptor agonist PD
168,077 also reduced the IPSCs, which was also associated
with paired-pulse facilitation. In addition, this agonist re-
duced the frequency of the mIPSCs with no effect on their
amplitude. The D4-receptor antagonist L-745,870 totally
blocked the effect of the D4-receptor agonist, indicating the
specificity of its effect. To verify the location of the receptors
on the pallidal terminals, these were eliminated by injecting
kainic acid into the globus pallidus. Kainic acid produced a
drastic (80%) fall in the globus pallidus neuronal population.
In this condition, the effect of the activation of D4 receptors
both on the IPSCs and mIPSCs was prevented, thus indicat-
ing that the location of the receptors was on the pallidal
terminals. Our results demonstrate that dopamine controls
the activity of the thalamic reticular neurons by regulating the
inhibitory input from the globus pallidus. © 2010 IBRO. Pub-
lished by Elsevier Ltd. All rights reserved.
Key words: basal ganglia, dopamine receptors, D4-agonists,
Parkinson’s disease, sleep disorders, thalamus.
The presence of markers of dopamine transmission in the
thalamus has been known for some time in rodents, pri-
mates, and humans (Wang et al., 1995; Freeman et al.,
2001; Sanchez-Gonzalez et al., 2005). In the rat, the do-
paminergic innervation of the thalamus originates from the
pars compacta of the substantia nigra (Freeman et al.,
2001; Prensa and Parent, 2001; Anaya-Martinez et al.,
2006). The innervation is particularly significant in the tha-
lamic reticular nucleus (TRn), as shown by the high density
of the dopamine transporter and the immunolabeling for
D1- and D4-dopamine receptors (Huang et al., 1992; Mr-
zljak et al., 1996; Khan et al., 1998; Freeman et al., 2001).
The TRn is made of a group of GABAergic neurons
(Houser et al., 1980; De Biasi et al., 1986) that modulate
the flow of information through the thalamus and that is
central for attention, waking, sleep, and the genesis of
various types of rhythmic activity (Steriade et al., 1985,
1986; Friedberg and Ross, 1993; von Krosigk et al., 1993;
McAlonan et al., 2008).
The TRn receives a significant GABAergic input from
the globus pallidus (external segment in primates) in rats
(Asanuma, 1989; Cornwall et al., 1990; Gandia et al.,
1993), and monkeys (Hazrati and Parent, 1991; Asanuma,
1994). In previous work (Floran et al., 2004a), we showed
that the D4-dopamine receptors modulate depolarization-
stimulated [3H]GABA release, which indicates the pres-
ence of the D4 receptors on GABAergic terminals.
Because neurons of the globus pallidus express D4-
dopamine receptors (Mrzljak et al., 1996; Ariano et al.,
1997), it is likely that the receptors are transported to the
axon terminals in the TRn. We have examined whether
the receptors are indeed present on the axon terminals
originating in the globus pallidus. We have studied the
effect of dopamine and D4-dopamine receptor agonists and
antagonists on inhibitory postsynaptic currents (IPSCs) and
miniature IPSCs (mIPSCs) on the TRn neurons. The
presence of receptors on pallidal terminals was shown
by measuring the disappearance of the effects of the
activation of the receptors after the lesion of the globus
pallidus with kainic acid. Preliminary results of this work
have been presented elsewhere (Gasca-Martinez et al.,
2009).
*Correspondence to: J. Aceves, Departamento de Fisiología, Biofísica
y Neurociencias, CINVESTAV-IPN, Apartado postal 14-740, México
D.F., 07000, México. Tel: ?52-55-5747-3800 Ext. 5188; fax: ?52-55-
5747-3754.
E-mail address: jaceves@fisio.cinvestav.mx (J. Aceves).
Abbreviations: ABC, avidin–biotin–peroxidase complex; ACSF, artifi-
cial cerebrospinal fluid; CPu, caudate–putamen; DAB, 3,3=-diamino-
benzidine tetrahydrochloride; EDS, excessive daytime sleepiness;
GP, globus pallidus; ic, internal capsule; IPSCs, inhibitory postsynaptic
currents; IT, low threshold-activation Ca2?current; L-745, L-745,870;
mIPSCs, miniature inhibitory postsynaptic currents; MPTP, 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine; PD, PD 168,077; REM, rapid eye
movement; Thal, thalamus; TRn, thalamic reticular nucleus; Vm, mem-
brane potential.
Neuroscience 169 (2010) 1672–1681
0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2010.05.048
1672

Page 2

EXPERIMENTAL PROCEDURES
Slice preparation and solutions
Experimental procedures were done in accordance with the Na-
tional Institutes of Health Guide for Care and Use of Laboratory
Animals and were approved by the Institutional Animal Care Com-
mittees of the CINVESTAV. Brain slices obtained from male
Wistar rats (postnatal day 14 to 21) were used. The rats were
anesthetized and decapitated. The brain was quickly removed
from the skull and placed in ice-cold artificial cerebrospinal fluid
(ACSF) containing (in mM): 124 NaCl, 26 NaHCO3, 2.5 KCl, 1.3
MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, and 10 glucose, pH 7.4 (with
95% O2and 5% CO2bubbling through the solution). To prevent
swelling of the cell during slicing, NaCl was replaced with choline
chloride. Both solutions were continuously oxygenated with the
gas mixture. Horizontal slices (300 ?m) containing the TRn (see
Fig. 1A) were cut with a vibroslicer (Lancer, Technical Products
International, St. Louis, MO, USA) and then transferred to normal
ACSF at room temperature (ca 25 °C) for equilibration. After 1 h,
a single slice was transferred to a recording chamber continuously
superfused with ACSF (1 to 2 mL/min) at room temperature. To
block N-methyl-D-aspartate and non-N-methyl-D-aspartate gluta-
mate receptors, AP-5 (50 ?M) and CNQX (10 ?M) were added to
the superfusion medium. TTX (1 ?M) was also added when study-
ing the effect of drugs on the mIPSCs.
Electrophysiology
Neurons were visualized using infrared differential-interference video
microscopy with a 40? water-immersion objective (Hamamatsu
C2400-50, Hamamatsu Photonics Systems, USA and Ax-
ioscop, Carl Zeiss, Oberkochen, Germany). Micropipettes for
whole cell recordings were pulled (Sutter Instruments, Novato,
CA, USA) from borosilicate glass tubes (1.5 mm outer diameter,
WPI, Sarasota, FL, USA) for a final resistance of 2 to 5 M? when
filled with a solution of the composition (in mM): 120 KSO3CH3, 16
KCl, 2 MgCl2, 10 HEPES, 1.1 K2-EGTA, 1.1 ATP-Mg, and 1.1
GTP-Na (pH 7.3 adjusted with KOH; osmolality, 287 to 290 mOsm
L?1). This internal solution was used for current-clamp recordings.
The IPSCs were recorded in a voltage-clamp condition (holding
potential??80 mV) with pipettes containing a solution of the
composition (in mM): 115 CsCl, 5 MgCl2, 10 HEPES, 10 K2-
EGTA, 4 ATP-Mg, and 1 GTP-Na. QX-314 (5 mM) was added to
avoid contamination of synaptic responses by unclamped action-
currents. The calculated chloride-equilibrium potential was 0 mV.
The IPSCs were produced using a bipolar, concentric Pt–Ir elec-
trode (50 ?m at the tip, 1 k? DC resistance; FHC, Bowdoinham,
ME, USA) at a frequency of 0.1 Hz using rectangular pulses (20
?s, 10 to 20 V; Digitimer Ltd isolated stimulator DS2, England).
The electrode was placed near (ca 100 ?m) the recorded cell. The
strength of the pulses was adjusted to produce synaptic currents
Fig. 1. Firing properties of the recorded neurons. (A) Location of the TRn in the brain slice. The asterisk indicates the location of the neuron shown
in (B). (B) Byocitin-filled neuron from which the recording was made. (C) At ?66 mV of the membrane potential, a depolarizing current pulse produces
tonic firing (upper trace); at ?88 mV, the same pulse produces a low-threshold calcium spike that triggers a burst of action potentials. (D) Illustration
of the ITcurrent recorded in another neuron. The lower trace shows that the current was eliminated by Ni2?(1 mM). Scale bars?400 ?m in (A); 27
?m in (B). ic, internal capsule; GP, globus pallidus; Thal, thalamus; TRn, thalamic reticular nucleus.
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811673

Page 3

of about 70% of the maximum amplitude. The paired-pulse pro-
tocol was done as previously described (Dunwiddie and Haas,
1985; Kamiya and Zucker, 1994). The interval between the pulses
was usually 100 ms. Voltage-clamp recordings were made with an
Axopatch 200A amplifier (Axon Instruments, Foster City, CA,
USA). Liquid junction potentials (?5 mV) were not corrected.
Recordings were acquired at 10 kHz using a Digidata 1200 inter-
face (Axon) and pCLAMP software (Axon, v 7.0). The Bessel filter
was set at 5 kHz. Access resistance (7 to 15 M?) was monitored
continuously and experiments were abandoned if changes ?20%
occurred. The low threshold-activation Ca2?current (IT) was ex-
plored in the presence of TTX to block Na?currents using a
voltage-clamp technique with a holding potential of ?50 mV.
Following conditioning hyperpolarization voltage steps (?50 to
?125 mV, 5 mV increments, 500 ms duration) with a step com-
mand to ?50 mV was used to produce the ITcurrent. Because D1
receptors are present in the TRn (Huang et al., 1992), the effects
of dopamine were studied in the presence of the D1-receptor
antagonist SCH 23390 (1 ?M) to rule out participation of this class
of receptors in the dopamine effects. To prevent dopamine oxida-
tion, ascorbic acid (1%) was always present in the bathing me-
dium.
Lesion of globus pallidus
Neonatal rats (postnatal day 7) were anesthetized by hypothermia
and placed on a home-made frame under a stereotaxic apparatus
(David Kopf), and 50 nL of kainic acid solution (1 ?g/?L freshly
dissolved in 0.1 M sodium phosphate buffer, pH 7.4) was injected
unilaterally into the globus pallidus (coordinates with respect to
bregma: lateral ?1.8 mm, anteroposterior ?0.7 mm, dorsoventral
?4.0 mm). The coordinates were previously determined by inject-
ing Methylene Blue. In all cases the injection site was located in
the central or medial part of the globus pallidus. One week after
the pallidal lesion was made, the rats were anesthetized and
perfused transcardially with 4% paraformaldehyde and 0.05%
gluteraldehyde in 0.1 M phosphate buffer, pH 7.4. The brain was
stored in this fixative diluted 50:50 with 20% sucrose at 4 °C for at
least 24 h. Then coronal 50-?m-thick slices were obtained. The
injection site was verified in alternate slices stained with Cresyl-
Violet. To determine the neuronal population, the neurons were
immunolabeled using a mouse monoclonal anti-NeuN (1:200;
Chemicon International, Temecula, CA, USA). In this latter case,
the slices were incubated for 24 h, then for 1 h in biotinylated
horse-antimouse secondary antibody. The slices were incubated
for 1 h in abidin–biotin peroxidase complex (Vectastain Elite ABC
kit), then for 10 min in 3,3=-diaminobenzidine tetrahydrochloride
(DAB). The slices were mounted on gelatine-coated slides, dried,
dehydrated, and coverslipped in DPX resin.
Drugs
D-(?)-2-amino-5-phosphonopentanoic acid (AP-5), 6-Cyano-7-ni-
troquinoxaline-2,3-dione (CNQX), bicuculline methiodide, N-[[4-
(2-cyanophenyl)-1-piperazinyl]methyl]-3-methyl-benzamide mal-
eate (PD 168,077), (3-[(4-[4-chlorophenyl]piperazin-1-yl)methyl]-
1H pyrrolo[2,3-b]pyridine) hydrochloride (L-745,870), lidocaine
N-ethyl bromide (QX-314), tetrodotoxin (TTX), (RS)-2,3,4,5 tetra-
hydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine hydrochloride
(SKF 38393), R(?)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,
5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390), 3,4-
dihydroxyphenethylamine (dopamine), and S-(?)-5-amino-sulfonyl-
N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide(sulpiride)were
obtained from Sigma (St. Louis, MO, USA). The drugs were stored
in the freezer as dry aliquots. Stock solutions were prepared just
before each experiment and added to the perfusion solution at the
final concentration.
Statistics
The data were analyzed using Prism 4 (Graph Pad) software. The
statistical significance of the differences of drug effects and
paired-pulse ratios was tested by the Wilcoxon test. The effects on
the mIPSCs were estimated by the Kolmogorov–Smirnov two-
sample test from amplitudes and cumulative inter-event interval
distributions (Minianalysis, Synaptosoft). The results are given as
means?SE with P?0.05 taken as a significant difference.
RESULTS
Electrophysiological properties of the recorded
neurons
Most recorded neurons were located on the ventrocaudal
part of the TRn (see Fig. 1A). Consistent with previous
reports (Jahnsen and Llinas, 1984a,b), the current injec-
tion at a depolarized Vm(?66 mV) produced a tonic dis-
charge. At hyperpolarized Vm(?88 mV), the same current
pulse produced a low-threshold calcium spike, which was
crowned by a burst of action potentials (Fig. 1C). These
firing properties have been consistently found by several
authors (Mulle et al., 1986; Spreafico et al., 1988; Con-
treras et al., 1992, 1993; Brunton and Charpak, 1997). Fig.
1B illustrates the neuron from which the recordings were
made. The neuron showed a dendritic arborization extend-
ing in the horizontal plane with an orientation parallel to the
axis of the nucleus, as those previously found in the TRn
(Mulle et al., 1986; Spreafico et al., 1988; Cox et al., 1996;
Pinault et al., 1997). Most of the recorded neurons pos-
sessed the ITcurrent (see Fig. 1D), which underlies the
calcium spike. The presence or absence of this current
was used to determine whether the neuron was of a tonic
or bursting type (Lee et al., 2007); 93% (n?60) of the
studied neurons showed the current, a value a little higher
than that reported by Lee et al. (2007), who reported 82%
(n?84).
Dopamine inhibits GABAergic IPSCs
The effect of dopamine on GABAergic transmission in the
TRn was studied on orthodromically stimulated IPSCs in the
presence of CNQX and AP-5. In this condition, the transmis-
sion was mediated by GABAAreceptors because it was
eliminated by bicuculline (not illustrated). As shown in Fig.
2A, exposure of the slices to dopamine (30 ?M) resulted in
about 40% inhibition of the amplitude of the IPSC (control,
327?34 pA; dopamine, 199?18 pA; P?0.001; n?9). The
inhibition did not modify the IPSC kinetics (see normalized
traces in Fig. 2B) and was associated with paired-pulse
facilitation (paired-pulse ratios: control, 0.99?0.05; dopa-
mine, 1.19?0.06; P?0.01; n?7) (Fig. 2C). These results
suggest a presynaptic location of the involved receptors.
Pharmacological profile of the D4 receptors that
mediate the inhibition
The effect of dopamine was dose-dependent. The maxi-
mum inhibitory response was obtained at a concentration
of 30 ?M (Fig. 3A). To identify the receptor responsible for
the dopamine inhibition, the action of various dopamine-
receptor antagonists was tested. The selective D4-recep-
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811674

Page 4

tor antagonist L-745,870 (L-745) partially blocked the effect
of dopamine (percent reduction of the IPSC: dopamine,
40?3; L-745 (1 ?M)?dopamine, 26?5; P?0.01; n?9).
The same reduction of the dopamine inhibition was ob-
tained by 30 ?M L-745 (Fig. 3B). To test a possible partic-
ipation of D2/D3 receptors in the dopamine inhibition,
sulpiride (50 ?M) was added concomitantly with dopamine.
As shown in Fig. 3B, the presence of sulpiride in the
medium did not prevent the inhibitory effect of dopamine,
ruling out any participation of the D2/D3 receptors in the
effect.
Dopamine reduces the frequency of the mIPSCs
In this series of experiments we explored whether dopa-
mine (30 ?M) affected the mIPSCs and whether the effect
was mediated by D4 receptors. As seen in Fig. 4A, in the
presence of 1 ?M L-745, dopamine had no effect on the
mIPSCs, but it reduced the frequency of the mIPSCs as
soon as the L-745 was removed from the bathing medium
(frequency (Hz): control, 0.78?0.1; L-745?dopamine,
0.74?0.1; dopamine, 0.46?0.06; P?0.01, dopamine vs.
control; amplitude (pA): control, 65?5; L-745?dopamine,
65?6; dopamine, 64?4; P?0.05; n?7). However, dopa-
mine had no effect on three out of 10 studied neurons. As
seen in the normalized traces of the inset, dopamine did
not affect the decay phase of the mIPSC. Dopamine re-
duced the frequency but did not affect the amplitude of the
mIPSCs (Fig. 4B). The results of seven experiments in
which L-745 showed effects are illustrated in the box plots
of Fig. 4C. The L-745 did not consistently block the effect of
dopamine and in five independent experiments L-745 did
not block the dopamine inhibition of the mIPSC frequency
(data not shown).
Because there is immunoreactivity for the D1-dopa-
mine receptors in the TRn (Huang et al., 1992), we tested
the effect of SKF 38393, a D1-like agonist, on the mIPSCs.
At 5 ?M, SKF 38393 had no effect either on the frequency
or the amplitude of the mIPSCs (frequency (Hz): control,
0.87?0.26; SKF 38393, 0.84?0.25; P?0.4; amplitude
(pA): control, 50.2?8.1; SKF 38393, 52?9.4; P?0.3; n?
7). For this series of experiments SCH 23390 was re-
moved from the bathing medium. Accordingly, it seems
that the D1-class of dopamine receptors does not affect
GABA transmission in the TRn.
Fig. 2. Dopamine reduces the IPSCs in TRn slices. (A) The time-course of the effect of dopamine (30 ?M). Inset shows the IPSCs recorded as
indicated in the time-course (a, b, and c). (B) Representative traces of dopamine action showing an increase in the paired-pulse ratio. The IPSCs were
normalized with respect to the first IPSC on the top trace. (C) Plot summarizing seven independent experiments showing the increase of the
paired-pulse ratio. * P?0.01 vs. control.
Fig. 3. The effect of dopamine is concentration-dependent. (A) Effect
of dopamine at progressively increasing concentrations. The maxi-
mum response was obtained at 30 ?M. (B) Effect of the D2/D3
receptors antagonist sulpiride and the D4-receptor antagonist
L-745,870 on the dopamine inhibition of the IPSCs (* P?0.01 vs.
dopamine). The numbers of experiments are shown in parentheses.
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811675

Page 5

Effect of the selective D4-agonist PD 168,077 on the
IPSCs and mIPSCs
To confirm the participation of the D4 receptors, the effect
of the selective D4-receptor agonist PD 168,077 (PD) was
explored. As seen in Fig. 5A, the agonist (1 ?M) reduced
the IPSC amplitude by 39% (control, 280?55 pA; PD,
171?36 pA; P?0.03 pA; n?7). To determine the specific-
ity of the effect of PD, its effect was first explored in the
presence of the selective D4-receptor antagonist L-745. As
seen in Fig. 5B, the antagonist totally blocked the effect of
the agonist. As soon as the antagonist was removed from
the medium, the effect of the agonist was revealed (con-
trol, 276?42 pA; L-745?PD, 283?75 pA; PD, 174?32 pA;
n?5; P?0.001, PD vs. control). The reduction in the IPSC
amplitude caused by PD was associated with a paired-
pulse facilitation (paired-pulse ratios: control, 0.93?0.04;
L-745?PD, 0.91?0.04; PD, 1.15?0.05; n?5; P?0.002,
PD vs. control) (see Fig. 5C, D). These results suggest a
presynaptic location of the receptors. The PD had no effect
in seven of 19 neurons. In contrast, dopamine effected the
IPSCs in all neurons tested (n?53).
The effect of PD was also tested on the mIPSCs. As
seen in Fig. 6, the D4 agonist reduced the frequency of the
mIPSCs without any effect on their amplitude. As seen in
the figure, the effect of PD was prevented by the selective
D4-receptor antagonist L-745, indicating the specificity of
the agonist effect (frequency (Hz): control, 0.67?0.04;
L-745?PD, 0.67?0.03; PD, 0.52?0.03; P?0.01, PD vs.
control; amplitude (pA): control, 47?1.3; L-745?PD, 46?
1.1; PD, 45?1.1; P?0.05; n?7). As for the IPSCs, there
were some neurons where the PD did not have any effect.
In five of 12 neurons, the D4 agonist did not reduce the
frequency of the mIPSCs.
The lesion of the globus pallidus eliminates the
inhibition produced by the D4 receptors
To determine the location of the D4 receptors, the neurons
of the globus pallidus were eliminated by injecting kainic
acid into the nucleus. The injection of kainic acid reduced
the neuronal population by about 80% (Fig. 7A). Fig. 7B
illustrates the results of one experiment in which the effect
of the activation of D4 receptors in the TRn was tested on
the side with the lesion compared to the intact side in slices
obtained from the same rat brain. It can be seen that the
lesion prevented the effect of the D4 agonist whereas
preserving its effect on the intact side. The same result
was observed in 10 additional experiments (inset, Fig. 7B).
In all of them the lesion prevented the D4-activation effect
(IPSC side with the lesion: control, 251?46 pA; PD,
255?47 pA; P?0.05; intact side: control, 364?65 pA; PD,
264?57 pA; P?0.01). Fig. 7C shows that the lesion elim-
inates the effect of the D4 agonist on the mIPSC frequency
(Hz): control, 0.58?0.1; PD, 0.55?0.1; P?0.1; amplitude
(pA): control, 44.5?5.6; PD, 44.5?5.4; P?0.9; n?8.
These results showed that indeed the D4 receptors located
on the pallidal terminals were responsible for the inhibition
of the GABAergic transmission.
DISCUSSION
Our main finding is that the GABAergic input from the
globus pallidus to the TRn is modulated by dopamine via
receptors located on the axon terminals of the palli-
doreticular projection and with a pharmacological profile of
D4 receptors. By activating these receptors, dopamine
reduces the inhibitory input to the TRn neurons. This is
consistent with a D4-receptor-mediated inhibition of depo-
larization-stimulated [3H]GABA release (Floran et al.,
2004a) from the TRn slices, with a dense immunoreactivity
for the D4-dopamine receptors (Mrzljak et al., 1996) and
the dopamine transporter (Freeman et al., 2001) in the
TRn, and with the dopamine innervation of the nucleus by
Fig. 4. Dopamine reversibly reduces, via the D4 receptors, the fre-
quency of the mIPSCs in TRn slices. (A) In the presence of the
D4-receptor antagonist L-745,870 (1 ?M) dopamine had no effect. The
effect of dopamine (30 ?M) was observed after removing the antago-
nist. The inset shows expanded traces of the mIPSCs (a–c). The
normalized traces showed that dopamine did not modify the mIPSCs
kinetics. (B) The cumulative distribution of the inter-event intervals and
amplitudes of the recordings is illustrated in (A). The Kolmogorov–
Smirnov test indicates the significance of the difference between do-
pamine vs. control. (C) Box plots summarizing the results of seven
experiments like the one shown in (A) and (B). * P?0.01 dopamine vs.
control.
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811676

Page 6

neurons from the pars compacta of the substantia nigra
(Anaya-Martinez et al., 2006).
The effect of dopamine was seen in most of the neu-
rons tested. Most neurons had ITcalcium currents, typical
of the bursting type of reticular neurons (Jahnsen and
Llinas, 1984a,b; Huguenard and Prince, 1992; Lee et al.,
2007). However, dopamine had effect independent of the
type of firing pattern of the neuron. Although the tested
neurons were located mostly on the ventrocaudal part of
the TRn, the dopamine modulation of the pallidal input is
probably exerted on most thalamic reticular neurons, be-
cause the immunoreactivity for D4 receptors (Mrzljak et al.,
1996) and for the dopamine transporter (Freeman et al.,
2001) labels the entire nucleus. However, there was some
small percentage of neurons in which the activation of the
D4 receptors did not affect the GABA transmission, sug-
gesting that not all pallidal neurons express the receptors.
In a previous work (Floran et al., 2004a), we showed that
D4-dopamine receptors inhibited depolarization-stimulated
[3H]GABA release in slices of the TRn, but the source of
the GABA release remained undetermined. The associa-
tion of paired-pulse facilitation with the dopamine inhibition
of the IPSCs and the dopamine-caused decrease of the
frequency of the mIPSCs, with no effect on the amplitude,
suggested a presynaptic location of the receptors. The
disappearance of the inhibition of the GABAergic transmis-
sion by activation of D4 receptors after forming a lesion on
the pallidal neurons showed that the location of the recep-
tors on terminals originated in the globus pallidus.
The projection from the globus pallidus to the TRn is
well-established both in monkeys (Hazrati and Parent,
1991; Asanuma, 1994) and rodents (Asanuma, 1989;
Cornwall et al., 1990; Gandia et al., 1993). The pallidal
terminals arborize profusely within the entire rostrocaudal
extent of the structure, ending mostly as large varicosities
closely apposed to cell bodies and proximal dendrites
(Asanuma, 1994; Parent and Hazrati, 1995), a topography
well suited to exert a powerful inhibitory control on the
activity of reticular neurons. By controlling the synaptic
strength of this input, dopamine may exert a significant
influence on the firing activity or firing patterns of reticular
neurons.
The expression of D4 receptors by neurons of the
globus pallidus of the rat has been shown by in situ hy-
bridization (Ariano et al., 1997), immunocytochemistry (Mr-
zljak et al., 1996; Khan et al., 1998), and electrophysiology
Fig. 5. The selective D4-receptor agonist PD 168,077 reversibly reduces the IPSC amplitude in the TRn slices. (A) The time-course of the effect of
PD 168,077 (1 ?M). Inset shows the IPSCs recorded as indicated in the time-course (a, b, and c). (B) The time-course of the effect of PD 168,077
(1 ?M) in the presence of the D4-receptor antagonist L-745,870 (1 ?M) and after its removal. Note that L-745,870 totally blocked the effect of PD
168,077. In the inset are traces obtained at (a, b, c, and d). (C) Representative traces illustrating that the effect of PD 168,077 was associated with
paired-pulse facilitation. The IPSCs were normalized with respect to the first IPSC on the top trace. (D) The paired–pulse facilitation obtained in five
independent experiments. * P?0.01 PD 168,077 vs. control.
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811677

Page 7

(Shin et al., 2003; Hernandez et al., 2006). The D4 recep-
tors located at somatic and dendritic membranes modulate
the inhibitory input from the striatum (Shin et al., 2003) and
the excitatory input from the subthalamic nucleus (Hernan-
dez et al., 2006).
Pallidal neurons send D4 receptors to the axon termi-
nals within the TRn (present results), subthalamic nucleus
(Floran et al., 2004b), and substantia nigra pars reticulata
(Acosta-Garcia et al., 2009). By activating these presyn-
aptic receptors dopamine reduces the inhibitory input from
the globus pallidus to the neurons of all these nuclei.
Interestingly, it appears that a single dopamine neuron of
the pars compacta of the substantia nigra may simulta-
neously activate the receptors located on all these different
pallidal terminals, because the same dopamine neuron
appears to innervate, via collaterals, all these nuclei
(Anaya-Martinez et al., 2006).
Possible physiopathological implications
It is well-established that sleep disturbances, excessive
daytime sleepiness (EDS), and rapid eye movement
(REM) sleep deregulation are frequently found in Parkin-
son’s disease (Rye et al., 2000; Arnulf et al., 2002; Arnulf,
2005). These disturbances may precede the motor alter-
ations of the disease by years and mark the disease early
(Schenck et al., 1996; Abbott et al., 2005; Happe et al.,
2007). Deregulation of the REM sleep and increased day-
time sleepiness occurring before the emergence of motor
symptoms has also been found in 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys (Bar-
raud et al., 2009). The presence of the sleep disorders in
the absence of motor alterations suggests that the loss of
dopamine in the caudate–putamen is not the cause of the
disorders. Because the TRn is the main factor determining
sleep–wake states (Steriade et al., 1986; McCormick and
Bal, 1997; Avanzini et al., 2000), it is tempting to speculate
that the loss of dopamine in the thalamus, in particular in
the TRn, may be the cause of the sleep disorders preced-
ing the motor manifestations of Parkinson’s disease. The
dopamine neurons innervating the TRn may degenerate
before the degeneration of those innervating the caudate–
putamen, which are the ones controlling motor behavior
(DeLong, 1990).
The major dysfunction after dopamine depletion ap-
pears to be the loss of functional segregation and the
appearance of oscillatory activity in corticobasal ganglia
circuits, which could cause most of the Parkinsonian
pathophysiology (Bar-Gad and Bergman, 2001; Wichmann
and DeLong, 2003; Gatev et al., 2006). Pessiglione et al.
(2005) did not find in MPTP-treated monkeys the drastic
inhibition of neuronal firing expected from rate models to
explain Parkinsonism (Albin et al., 1989; DeLong, 1990),
instead they found that the Parkinsonian state did not
change either the firing rate or pattern of the thalamic
neurons. They also found increased correlation in the firing
between a pair of neurons in the thalamic nuclei receiving
the projections from the basal ganglia. There was also a
loss of the specificity of the response to somatosensory
stimulation. Because the TRn neurons process sensory
information and trigger synchronized oscillatory activity
(Steriade et al., 1985; von Krosigk et al., 1993; Kim et al.,
1997; Bazhenov et al., 1999), it seems reasonable to
speculate that the decrease in specificity and the increase
in correlated firing could be, at least in part, associated with
the loss of the dopamine control of the inhibitory pallidal
input to the TRn neurons.
The end result of the dopaminergic modulation of the
pallidal input to the TRn is unknown. By reducing the tonic
GABAergic input from the globus pallidus, dopamine may
cause depolarization of the cells thereby favoring waking
and alertness (Steriade et al., 1986, 1993; McCormick and
Bal, 1997). However, this point should be explored in detail
to understand the role of dopamine modulating the activity
of thalamic reticular neurons.
In summary, our results may help the understanding of
the role of dopamine in the control of motor activity and
Fig. 6. The selective D4-agonist PD 168,077 reduces the frequency of
the mIPSCs. (A) In the presence of L-745,870 (1 ?M), the D4-receptor
antagonist PD 168,077 (1 ?M) had no effect. The effect of the PD
168,077 was observed after removing the antagonist. The inset shows
expanded traces of the mIPSCs (a–c). (B) The cumulative distribution
of the inter-event intervals and amplitudes of the recordings illustrated
in (A). The Kolmogorov–Smirnov test indicates the significance of the
difference between PD 168,077 vs. control. (C) Box plots summarizing
the results of seven experiments like the one shown in (A) and (B). *
P?0.01 PD 168,077 vs. control.
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811678

Page 8

sleep–wake states in physiological and pathological con-
ditions, such as Parkinson’s disease.
Acknowledgments—The technical assistance of Valentina Ramirez
Rosas and Lidia Jimenez Peña. This work was supported by a grant
(50427Q) from CONACyT (México) to J. Aceves. D. Gasca-Mar-
tinez was recipient of a fellowship from CONACyT. Thanks to Dr.
Ellis Glazier for editing this English-language text.
REFERENCES
Abbott RD, Ross GW, White LR, Tanner CM, Masaki KH, Nelson JS,
Curb JD, Petrovitch H (2005) Excessive daytime sleepiness and
subsequent development of Parkinson disease. Neurology 65:
1442–1446.
Acosta-Garcia J, Hernandez-Chan N, Paz-Bermudez F, Sierra A, Erlij
D, Aceves J, Floran B (2009) D4 and D1 dopamine receptors
modulate [3H] GABA release in the substantia nigra pars reticulata
of the rat. Neuropharmacology 57:725–730.
Albin RL, Young AB, Penney JB (1989) The functional anatomy of
basal ganglia disorders. Trends Neurosci 12:366–375.
Anaya-Martinez V, Martinez-Marcos A, Martinez-Fong D, Aceves J,
Erlij D (2006) Substantia nigra compacta neurons that innervate
the reticular thalamic nucleus in the rat also project to striatum or
globus pallidus: implications for abnormal motor behavior. Neuro-
science 143:477–486.
Fig. 7. The lesion of the neurons of the globus pallidus eliminates the inhibitory effect of the D4 receptors on the IPSCs and mIPSCs in the TRn.
(A) The left picture shows a coronal slice showing that kainic acid was injected into the globus pallidus (arrowhead). The middle picture shows the
drastic reduction (about 80%) in the neuronal population produced by the injection of kainic acid compared with the intact side from the same rat (right
picture). (B) This illustrates the effect of PD 168,077 (1 ?M) on the IPSCs in the TRn of the side with the lesion and the intact side of the same rat.
The upper insets show the results of 10 experiments. It is clear that the lesion eliminated the effect of the activation of the D4 receptors. (C)
Representative traces showing that the lesion also eliminated the effect of the activation of D4 receptors on the frequency of the mIPSCs. Bicuculline
eliminated the mIPSCs, indicating that they were still mediated by GABAAreceptors. The box plots summarize the results of eight independent
experiments. CPu, caudate–putamen; ic, internal capsule; GP, globus pallidus.
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811679

Page 9

Ariano MA, Wang J, Noblett KL, Larson ER, Sibley DR (1997) Cellular
distribution of the rat D4 dopamine receptor protein in the CNS
using anti-receptor antisera. Brain Res 752:26–34.
Arnulf I, Konofal E, Merino-Andreu M, Houeto JL, Mesnage V, Welter
ML, Lacomblez L, Golmard JL, Derenne JP, Agid Y (2002) Parkin-
son’s disease and sleepiness: an integral part of PD. Neurology
58:1019–1024.
Arnulf I (2005) Excessive daytime sleepiness in parkinsonism. Sleep
Med Rev 9:185–200.
Asanuma C (1989) Axonal arborizations of a magnocellular basal
nucleus input and their relation to the neurons in the thalamic
reticular nucleus of rats. Proc Natl Acad Sci U S A 86:4746–4750.
Asanuma C (1994) GABAergic and pallidal terminals in the thalamic
reticular nucleus of squirrel monkeys. Exp Brain Res 101:
439–451.
Avanzini G, Panzica F, de Curtis M (2000) The role of the thalamus in
vigilance and epileptogenic mechanisms. Clin Neurophysiol 111
(Suppl 2):S19–S26.
Bar-Gad I, Bergman H (2001) Stepping out of the box: information
processing in the neural networks of the basal ganglia. Curr Opin
Neurobiol 11:689–695.
Barraud Q, Lambrecq V, Forni C, McGuire S, Hill M, Bioulac B,
Balzamo E, Bezard E, Tison F, Ghorayeb I (2009) Sleep disorders
in Parkinson’s disease: the contribution of the MPTP non-human
primate model. Exp Neurol 219:574–582.
Bazhenov M, Timofeev I, Steriade M, Sejnowski TJ (1999) Self-sus-
tained rhythmic activity in the thalamic reticular nucleus mediated
by depolarizing GABAA receptor potentials. Nat Neurosci 2:
168–174.
Brunton J, Charpak S (1997) Heterogeneity of cell firing properties and
opioid sensitivity in the thalamic reticular nucleus. Neuroscience
78:303–307.
Contreras D, Curro Dossi R, Steriade M (1992) Bursting and tonic
discharges in two classes of reticular thalamic neurons. J Neuro-
physiol 68:973–977.
Contreras D, Curro Dossi R, Steriade M (1993) Electrophysiological
properties of cat reticular thalamic neurones in vivo. J Physiol
470:273–294.
Cornwall J, Cooper JD, Phillipson OT (1990) Projections to the rostral
reticular thalamic nucleus in the rat. Exp Brain Res 80:157–171.
Cox CL, Huguenard JR, Prince DA (1996) Heterogeneous axonal
arborizations of rat thalamic reticular neurons in the ventrobasal
nucleus. J Comp Neurol 366:416–430.
De Biasi S, Frassoni C, Spreafico R (1986) GABA immunoreactivity in
the thalamic reticular nucleus of the rat. A light and electron mi-
croscopical study. Brain Res 399:143–147.
DeLong MR (1990) Primate models of movement disorders of basal
ganglia origin. Trends Neurosci 13:281–285.
Dunwiddie TV, Haas HL (1985) Adenosine increases synaptic facili-
tation in the in vitro rat hippocampus: evidence for a presynaptic
site of action. J Physiol 369:365–377.
Floran B, Floran L, Erlij D, Aceves J (2004a) Activation of dopamine D4
receptors modulates [3H]GABA release in slices of the rat thalamic
reticular nucleus. Neuropharmacology 46:497–503.
Floran B, Floran L, Erlij D, Aceves J (2004b) Dopamine D4 receptors
inhibit depolarization-induced [3H]GABA release in the rat subtha-
lamic. Eur J Pharmacol 498:97–102.
Freeman A, Ciliax B, Bakay R, Daley J, Miller RD, Keating G, Levey A,
Rye D (2001) Nigrostriatal collaterals to thalamus degenerate in
parkinsonian animal models. Ann Neurol 50:321–329.
Friedberg EB, Ross DT (1993) Degeneration of rat thalamic reticular
neurons following intrathalamic domoic acid injection. Neurosci
Lett 151:115–119.
Gandia JA, De Las Heras S, Garcia M, Gimenez-Amaya JM (1993)
Afferent projections to the reticular thalamic nucleus from the glo-
bus pallidus and the substantia nigra in the rat. Brain Res Bull
32:351–358.
Gasca-Martinez D, Hernandez A, Sierra A, Anaya-Martinez V, Valdi-
osera R, Floran B, Erlij D, Aceves J (2009) Dopamine reduces the
GABAergic input from globus pallidus to thalamic reticular nucleus
via presynaptic D4 dopamine receptors. Soc Neurosci Abstr
566.11/EE26.
Gatev P, Darbin O, Wichmann T (2006) Oscillations in the basal
ganglia under normal conditions and in movement disorders. Mov
Disord 21:1566–1577.
Happe S, Baier PC, Helmschmied K, Meller J, Tatsch K, Paulus W
(2007) Association of daytime sleepiness with nigrostriatal dopa-
minergic degeneration in early Parkinson’s disease. J Neurol
254:1037–1043.
Hazrati LN, Parent A (1991) Projection from the external pallidum to
the reticular thalamic nucleus in the squirrel monkey. Brain Res
550:142–146.
Hernandez A, Ibanez-Sandoval O, Sierra A, Valdiosera R, Tapia D,
Anaya V, Galarraga E, Bargas J, Aceves J (2006) Control of the
subthalamic innervation of the rat globus pallidus by D2/3 and D4
dopamine receptors. J Neurophysiol 96:2877–2888.
Houser CR, Vaughn JE, Barber RP, Roberts E (1980) GABA neurons
are the major cell type of the nucleus reticularis thalami. Brain Res
200:341–354.
Huang Q, Zhou D, Chase K, Gusella JF, Aronin N, DiFiglia M (1992)
Immunohistochemical localization of the D1 dopamine receptor in rat
brain reveals its axonal transport, pre- and postsynaptic localization,
and prevalence in the basal ganglia, limbic system, and thalamic
reticular nucleus. Proc Natl Acad Sci U S A 89:11988–11992.
Huguenard JR, Prince DA (1992) A novel T-type current underlies
prolonged Ca(2?)-dependent burst firing in GABAergic neurons of
rat thalamic reticular nucleus. J Neurosci 12:3804–3817.
Jahnsen H, Llinas R (1984a) Electrophysiological properties of guinea-
pig thalamic neurones: an in vitro study. J Physiol 349:205–226.
Jahnsen H, Llinas R (1984b) Ionic basis for the electro-responsive-
ness and oscillatory properties of guinea-pig thalamic neurones in
vitro. J Physiol 349:227–247.
Kamiya H, Zucker RS (1994) Residual Ca2? and short-term synaptic
plasticity. Nature 371:603–606.
Khan ZU, Gutierrez A, Martin R, Penafiel A, Rivera A, De La Calle A
(1998) Differential regional and cellular distribution of dopamine D2-
like receptors: an immunocytochemical study of subtype-specific an-
tibodies in rat and human brain. J Comp Neurol 402:353–371.
Kim U, Sanchez-Vives MV, McCormick DA (1997) Functional dynam-
ics of GABAergic inhibition in the thalamus. Science 278:130–134.
Lee SH, Govindaiah G, Cox CL (2007) Heterogeneity of firing proper-
ties among rat thalamic reticular nucleus neurons. J Physiol
582:195–208.
McAlonan K, Cavanaugh J, Wurtz RH (2008) Guarding the gateway to
cortex with attention in visual thalamus. Nature 456:391–394.
McCormick DA, Bal T (1997) Sleep and arousal: thalamocortical
mechanisms. Annu Rev Neurosci 20:185–215.
Mrzljak L, Bergson C, Pappy M, Huff R, Levenson R, Goldman-Rakic
PS (1996) Localization of dopamine D4 receptors in GABAergic
neurons of the primate brain. Nature 381:245–248.
Mulle C, Madariaga A, Deschenes M (1986) Morphology and electro-
physiological properties of reticularis thalami neurons in cat: in vivo
study of a thalamic pacemaker. J Neurosci 6:2134–2145.
Parent A, Hazrati LN (1995) Functional anatomy of the basal ganglia.
II. The place of subthalamic nucleus and external pallidum in basal
ganglia circuitry. Brain Res Brain Res Rev 20:128–154.
Pessiglione M, Guehl D, Rolland AS, Francois C, Hirsch EC, Feger J,
Tremblay L (2005) Thalamic neuronal activity in dopamine-de-
pleted primates: evidence for a loss of functional segregation within
basal ganglia circuits. J Neurosci 25:1523–1531.
Pinault D, Smith Y, Deschenes M (1997) Dendrodendritic and axoax-
onic synapses in the thalamic reticular nucleus of the adult rat.
J Neurosci 17:3215–3233.
Prensa L, Parent A (2001) The nigrostriatal pathway in the rat: a
single-axon study of the relationship between dorsal and ventral
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811680

Page 10

tier nigral neurons and the striosome/matrix striatal compartments.
J Neurosci 21:7247–7260.
Rye DB, Bliwise DL, Dihenia B, Gurecki P (2000) FAST TRACK:
daytime sleepiness in Parkinson’s disease. J Sleep Res 9:63–69.
Sanchez-Gonzalez MA, Garcia-Cabezas MA, Rico B, Cavada C
(2005) The primate thalamus is a key target for brain dopamine.
J Neurosci 25:6076–6083.
Schenck CH, Bundlie SR, Mahowald MW (1996) Delayed emergence
of a parkinsonian disorder in 38% of 29 older men initially diag-
nosed with idiopathic rapid eye movement sleep behaviour disor-
der. Neurology 46:388–393.
Shin RM, Masuda M, Miura M, Sano H, Shirasawa T, Song WJ,
Kobayashi K, Aosaki T (2003) Dopamine D4 receptor-induced
postsynaptic inhibition of GABAergic currents in mouse globus
pallidus neurons. J Neurosci 23:11662–11672.
Spreafico R, de Curtis M, Frassoni C, Avanzini G (1988) Electrophys-
iological characteristics of morphologically identified reticular tha-
lamic neurons from rat slices. Neuroscience 27:629–638.
Steriade M, Deschenes M, Domich L, Mulle C (1985) Abolition of
spindle oscillations in thalamic neurons disconnected from nucleus
reticularis thalami. J Neurophysiol 54:1473–1497.
Steriade M, Domich L, Oakson G (1986) Reticularis thalami neurons
revisited: activity changes during shifts in states of vigilance.
J Neurosci 6:68–81.
Steriade M, McCormick DA, Sejnowski TJ (1993) Thalamocortical
oscillations in the sleeping and aroused brain. Science 262:
679–685.
von Krosigk M, Bal T, McCormick DA (1993) Cellular mechanisms of
a synchronized oscillation in the thalamus. Science 261:361–364.
Wang GJ, Volkow ND, Fowler JS, Ding YS, Logan J, Gatley SJ,
MacGregor RR, Wolf AP (1995) Comparison of two pet radioli-
gands for imaging extrastriatal dopamine transporters in human
brain. Life Sci 57:PL187–PL191.
Wichmann T, DeLong MR (2003) Pathophysiology of Parkinson’s
disease: the MPTP primate model of the human disorder. Ann N Y
Acad Sci 991:199–213.
(Accepted 21 May 2010)
(Available online 16 June 2010)
D. Gasca-Martinez et al. / Neuroscience 169 (2010) 1672–16811681