1Mental Retardation Research Center, University of California, Los Angeles, California 90095; 2Department of Cell and Developmental Biology and 3Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201; 4Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas and Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina; and 5Department of Neuroscience, The Chicago Medical School, North Chicago, Illinois 60064
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ABSTRACT |
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Cepeda, C., R. S. Hurst, K. L. Altemus, J. Flores-Hernández, C. R. Calvert, E. S. Jokel, D. K. Grandy, M. J. Low, M. Rubinstein, M. A. Ariano, and M. S. Levine. Facilitated Glutamatergic Transmission in the Striatum of D2 Dopamine Receptor-Deficient Mice. J. Neurophysiol. 85: 659-670, 2001. Dopamine (DA) receptors play an important role in the modulation of excitability and the responsiveness of neurons to activation of excitatory amino acid receptors in the striatum. In the present study, we utilized mice with genetic deletion of D2 or D4 DA receptors and their wild-type (WT) controls to examine if the absence of either receptor subtype affects striatal excitatory synaptic activity. Immunocytochemical analysis verified the absence of D2 or D4 protein expression in the striatum of receptor-deficient mutant animals. Sharp electrode current- and whole cell patch voltage-clamp recordings were obtained from slices of receptor-deficient and WT mice. Basic membrane properties were similar in D2 and D4 receptor-deficient mutants and their respective WT controls. In current-clamp recordings in WT animals, very little low-amplitude spontaneous synaptic activity was observed. The frequency of these spontaneous events was increased slightly in D2 receptor-deficient mice. In addition, large-amplitude depolarizations were observed in a subset of neurons from only the D2 receptor-deficient mutants. Bath application of the K+ channel blocker 4-aminopyridine (100 µM) and bicuculline methiodide (10 µM, to block synaptic activity due to activation of GABAA receptors) markedly increased spontaneous synaptic activity in receptor-deficient mutants and WTs. Under these conditions, D2 receptor-deficient mice displayed significantly more excitatory synaptic activity than their WT controls, while there was no difference between D4 receptor-deficient mice and their controls. In voltage-clamp recordings, there was an increase in frequency of spontaneous glutamate receptor-mediated inward currents without a change in mean amplitude in D2 receptor-deficient mutants. In WT mice, activation of D2 family receptors with quinpirole decreased spontaneous excitatory events and conversely sulpiride, a D2 receptor antagonist, increased activity. In D2 receptor-deficient mice, sulpiride had very little net effect. Morphologically, a subpopulation of medium-sized spiny neurons from D2 receptor-deficient mice displayed decreased dendritic spines compared with cells from WT mice. These results provide evidence that D2 receptors play an important role in the regulation of glutamate receptor-mediated activity in the corticostriatal or thalamostriatal pathway. These receptors may function as gatekeepers of glutamate release or of its subsequent effects and thus may protect striatal neurons from excessive excitation.
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INTRODUCTION |
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Dopamine (DA) receptors play an important role in the modulation
of excitability and the responsiveness of neurons to excitatory and
inhibitory amino acids in the striatum (Calabresi et al. 1997a, 2000
; Cepeda and Levine 1998
; Konradi et
al. 2000
; Levine and Cepeda 1998
). The effects
of DA are mediated by at least five identified receptor subtypes
classified into two families according to their pharmacological
profiles (Civelli et al. 1991
; Creese and Fraser
1997
; Sibley and Monsma 1992
). This
classification scheme recognizes a D1 family composed of
D1A and D1B subtypes in rat
or their corresponding D1 and
D5 forms in human, and a D2 family composed of
three subtypes, D2, D3, and
D4. To minimize confusion, we will use D1 and D2
to refer to the families of DA receptors and subscript notation (e.g.,
D2 and D4) to refer to subtypes within each family.
The effects of DA on the excitability of striatal cells are complex.
The outcome of DA modulation depends on a number of factors such as the
DA receptor subtype preferentially activated, the receptor location at
pre- or postsynaptic sites, the concentration of ambient DA, and the
activity-state of the neuron subject to DA modulation (Cepeda
and Levine 1998). We have proposed a relatively simple scheme
to explain some of DA's modulatory actions in which its effects are a
function of the specific receptor subtypes with which it interacts
(either the DA receptor subtypes or the neurotransmitter receptors that
it will modulate). In the striatum, D1 receptor activation enhances
responses elicited by glutamate receptors, particularly those mediated
by N-methyl-D-aspartate (NMDA) receptors. In
contrast, D2 receptor activation reduces glutamate receptor responses,
particularly those mediated by activation of non-NMDA receptors. The
enhancing effects of D1 receptor activation appear to involve
postsynaptic actions (Cepeda et al. 1993
, 1998a
;
Flores-Hernández et al. 1999
). The attenuating
effects mediated by D2 receptors may involve both presynaptic actions
on corticostriatal terminals and/or postsynaptic actions (Cepeda
et al. 1993
; Flores-Hernández et al. 1997
;
Levine et al. 1996b
).
Although there is considerable evidence for the importance of
functional D2 receptors in the striatum, their electrophysiological roles and their cellular location still remain areas of debate. One
major controversial area is whether D2 receptors are located presynaptically on corticostriatal axons regulating glutamate release
(Schwarcz et al. 1978) or are located postsynaptically on striatal neurons regulating excitability. Pharmacological studies have been the most consistent in supporting presynaptic DA receptor regulation of glutamate release via D2 receptors (Godukhin et al. 1984
; Kornhuber and Kornhuber 1986
;
Maura et al. 1988
, 1989
; Mitchell and Dogett
1980
; Rowlands and Roberts 1980
; Yamamoto and Davy 1992
). Studies examining receptor binding and
anatomical localization have provided conflicting outcomes. In some
cases, the existence of presynaptic D2 receptors was supported, whereas in others it was not (Fisher et al. 1994
; Garau
et al. 1978
; Hersch et al. 1995
; Joyce
and Marshall 1987
; Schwarcz et al. 1978
;
Sesack et al. 1994
; Trugman et al. 1986
).
Electrophysiological studies demonstrating pre- or postsynaptic actions
of D2 family receptors have also been inconclusive. In the first
studies examining effects of DA, both pre- and postsynaptic mechanisms
were implicated in the reduction of striatal excitability (Arbuthnott et al. 1987; Brown and Arbuthnott
1983
; Garcia-Muñoz et al. 1991
;
Mercuri et al. 1985
). In vitro experiments initially suggested that the reduction of excitatory postsynaptic potentials by
DA was mediated exclusively by postsynaptic D1 receptors
(Calabresi et al. 1987a
). D2 receptor-mediated effects
were not observed unless these receptors were rendered hypersensitive
by DA-depleting lesions (Calabresi et al. 1988
, 1993
).
Subsequent studies have revealed that D2 receptor-mediated effects were
not always dependent on rendering these receptors hypersensitive, and
electrophysiological evidence has been obtained for both pre- and
postsynaptic actions of D2 receptors in the striatum (Cepeda et
al. 1994
; Hsu et al. 1995
; Jiang and
North 1991
).
Another approach to examine DA's role in presynaptic modulation of
glutamatergic activity involves use of the K+
channel blocker 4-aminopyridine (4-AP) (Flores-Hernández
et al. 1994). This blocker causes release of excitatory and
inhibitory neurotransmitters. Addition of bicuculline (BIC), a
GABAA receptor antagonist, isolates excitatory
synaptic activity. Under these conditions, D2 receptor activation
reduces excitatory synaptic activity in a subset of neurons, probably
via presynaptic modulation (Flores-Hernández et al.
1997
).
The reasons for the inconsistencies in electrophysiological findings
probably stem from differences in experimental approach and recording
conditions. For example, modulation by D2 receptors in vitro has been
more difficult to demonstrate than in vivo (Brown and Arbuthnott
1983; Calabresi et al. 1996
; however, see
Hsu et al. 1995
). On the other hand, acute effects of DA
mediated by activation of D2 receptors are mild at best (Cepeda
et al. 1994
; Flores-Hernández et al. 1994
)
but can be enhanced after chronic DA depletion (Calabresi et al.
1988
, 1993
), hence the conclusion that the control of glutamate
release by presynaptic D2 receptors might operate in vivo under
physiological conditions, whereas in vitro it requires supersensitivity
of D2 receptors (Calabresi et al. 1996
). One overriding
factor that might help unravel the inconsistencies is examining
differences between acute effects of application of D2 receptor
agonists and antagonists and the more chronic effects of long-term
alterations in receptor function like pharmacological blockade or
lesions of DA inputs. The present experiments address these differences
by examining chronic effects of genetic deletion of specific DA
receptors as well as acute treatment with agonists and antagonists.
The recent generation of DA receptor-deficient mutant mice has provided
another tool to examine hypotheses concerning the specific role of each
DA receptor subtype using electophysiological analyses (Levine
et al. 1996a). In the present studies, we used D2 and D4 DA
receptor-deficient mutant mice to assess whether the removal of these
receptor subtypes alters DA's ability to modulate glutamate synaptic
responses in the striatum. Although D2 receptors
have generally been hypothesized as the prominent D2 receptor localized
in the striatum, the D4 receptor has also been
localized on corticostriatal terminals (Tarazi et al.
1998
) as well as on striatal neurons (Ariano et
al. 1997
). In the present study, we utilized intracellular and
whole cell patch-clamp recording techniques to examine spontaneous
depolarizations (current clamp with sharp electrodes) and spontaneous
excitatory synaptic currents (voltage clamp with patch electrodes) in
slices from D2 and D4 DA
receptor-deficient mutant mice. We hypothesized that if
D2 receptors are responsible for inhibitory
regulation of glutamate responses in the striatum, then there should be
an increase in spontaneous synaptic activity in the striatum in
D2 receptor-deficient mice. If, on the other
hand, D4 receptors are responsible for inhibitory
regulation, then there should be an increase in spontaneous synaptic
activity in D4 receptor-deficient mice.
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METHODS |
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Animals
D2 and D4
receptor-deficient mutant mice were obtained from the colony of Dr.
David K. Grandy at the Oregon Health Sciences University, Portland, OR.
The methods used for the generation and identification of these mice
have been described (Kelly et al. 1997, 1998
;
Rubinstein et al. 1997
). The D2
mice (129S4/SvEv; C57BL/6) were incipient congenics after five
backcrosses to C57BL/6J and the D4 mice
(129P2/OlaHsd, C57BL/6) were F2 hybrids. All
experimental procedures were carried out in accordance with the
National Institutes of Health Guide for Care and Use of Laboratory
Animals and were approved by the Institutional Animal Care and Use
Committee at UCLA. The total number of mice used in these experiments
was 60 [20 D2 receptor-deficient, 18 wild-type
(WT) littermates, 12 D4 receptor-deficient, 10 WT
littermates]. Ages ranged from 3 mo to up to 1 yr. For whole cell
recordings with patch electrodes, young animals (3 mo) were used
because visualization of individual neurons with infrared
videomicroscopy and the quality of the patches were better than in
older animals. For intracellular recordings with sharp electrodes and
anatomical studies, we had no age limitations and animals spanned the
range of 3 mo to 1 yr. There were no consistent differences in data due
to age, and results were pooled according to genotype.
Preparation of slices
Our procedures for brain slice preparation have been described
(Cepeda et al. 1998a; Levine et al.
1996a
,b
). Briefly, mice were anesthetized with halothane and
then euthanized by decapitation. After dissection, brains were placed
in ice-cold oxygenated artificial cerebrospinal fluid (ACSF) [which
contained (in mM) 130 NaCl, 26 NaHCO3, 3 KCl, 5 MgCl2, 1.25 NaH2PO4, 1.0 CaCl2, and 10 glucose 10 (pH 7.2-7.4)].
Striatal sections were cut (350 µm) in the coronal plane and placed
in oxygenated (95% O2-5%
CO2) ACSF (same as preceding except 2 mM
CaCl2 and 2 mM MgCl2).
After at least 1 h the slice was transferred to a Haas-type
recording chamber for standard intracellular recordings. For patch
recordings from visualized neurons, the slice was placed in a perfusion
chamber attached to the fixed-stage of an upright microscope (Zeiss
Axioskop) and submerged in continuously flowing oxygenated ACSF
(25°C, 4 ml/min). Cells were visualized with a 40× water-immersion
lens and illuminated with near infrared (IR) light (790 nm, Ealing
Optics, Hollston, MA), and the image was detected with an IR-sensitive
CCD camera. Cells were typically visualized from 30 to 100 µm below
the surface of the slice. It was possible to distinguish medium- and
large-sized striatal cells. In the present study, only medium-sized
neurons were examined.
Current-clamp recordings
To evaluate electrophysiological properties, standard
current-clamp recordings were obtained using high-impedance sharp
electrodes (3 M K-acetate, 80-120 M). Signals were amplified
(Axoclamp-2A, Axon Instruments, Foster City, CA), displayed on an
oscilloscope, and digitized for subsequent computer analysis (pClamp
6.0.1, Axon Instruments). All data were obtained from neurons with
resting membrane potentials (RMP) of at least
60 mV and action
potentials exceeding 55 mV. Membrane properties (RMP, action potential
amplitude and duration, input resistance, and rheobase) were measured.
Whole cell voltage clamp
The whole cell patch technique was used for voltage-clamp
recordings. Patch electrodes (3-6 M) were filled with the following internal solution (in mM): 130 Cs-methanesulfonate, 10 CsCl, 4 NaCl, 1 MgCl2, 5 MgATP, 5 EGTA, 10 HEPES, 0.5 GTP, 10 phosphocreatine, and 0.1 leupeptin (pH 7.25-7.3, osmolality 280-290
mOsm). Axopatch 200A or 1D amplifiers were used for voltage-clamp
recordings. A 3 M KCl agar bridge was inserted between the
extracellular solution and the Ag-AgCl indifferent electrode. Tight
seals (2-10 G
) from visualized medium-sized cells were obtained by
applying negative pressure. The membrane was disrupted with additional
suction and the whole cell configuration was obtained. The access
resistances ranged from 8 to 15 M
and were compensated 60-85%.
Cells were held at
60 mV.
Drug application
The K+ channel blocker 4-AP (100 µM) was
added to the ACSF to induce spontaneous synaptic activity
(Flores-Hernández et al. 1994). BIC (10 µM) was
used to block spontaneous activity caused by activation of
GABAA receptors. In these conditions, the
remaining activity was due to activation of glutamate receptors. A DA
D2 agonist (quinpirole, 10-20 µM) or
antagonist (sulpiride, 10 µM) was used to study the modulation of
4-AP-induced synaptic activity. Spontaneous or evoked synaptic activity
was analyzed using the Mini Analysis Program (Synaptosoft, Leonia, NJ).
Only events clearly distinguishable from background noise, usually
larger than 3 mV (current clamp) or 5 pA (voltage clamp) were included
in the analysis.
Cell identification
In all experiments, sharp electrodes were filled with 2%
biocytin and patch electrodes were filled with 0.2% biocytin (Sigma, St Louis, MO) to label neurons. After the experiment, the slice was
fixed in 4% paraformaldehyde overnight and then processed according to
published protocols (Kita and Armstrong 1991). For the
morphological analysis, only cells obtained from sharp electrode intracellular recordings were examined. Recovered neurons from experiments using patch electrodes were only identified as medium-sized spiny cells. Their morphology or the existence of dye-coupling was not
examined in these cells because of the possibility of dye leakage when
the seal was made.
Immunohistochemistry
Anti-peptide antisera developed for the D2
(McVittie et al. 1991) and D4
(Ariano et al. 1997
) receptor subtypes were used to
evaluate receptor expression in the D2 and
D4 receptor-deficient mice. Fresh-frozen brain
sections (10 µm thick) were cut in the coronal plane. Tissue sections
from receptor-deficient and WT control mice were processed
simultaneously to minimize potential differences in experimental
processing. The antisera were applied to the slide-mounted section and
incubated overnight at 4°C in a humidified environment. The following
day, unbound primary anti-DA receptor antisera were rinsed off and
secondary, fluorescently labeled anti-rabbit antisera were applied for
1-2 h at 4°C in a humidified environment. Sections were examined
immediately using a scanning laser confocal microscope (BioRad MRC
600). Digitized images for control and mutant striata were matched from
similar regions of the nucleus and obtained by 10 accumulated scans
from the laser without any further image enhancement. Laser settings were identical for each antiserum detected in an experimental pair.
Controls for the procedure included use of multiple antisera directed
against different epitopes of the receptor protein sequence, use of
preimmune sera, omission of primary antisera, and adsorption of the
primary antisera with the peptide antigen.
Statistics
Differences between group means were analyzed with t-tests or the appropriate nonparametric statistic. In the text and tables, values are presented as mean ± SE. Differences between means were considered statistically significant when P < 0.05.
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RESULTS |
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Immunohistochemistry
D2 receptor expression was examined in four
D2 receptor-deficient and four WT mice. Receptor
protein staining was prevalent within medium- and large-diameter
neurons in WT striata (Fig. 1, top
left) while immunofluorescence was absent in the
receptor-deficient mice (Fig. 1, top right). The cerebral
cortex of WT animals also showed moderate levels of receptor
expression. Receptor expression for D4 receptors
was unaltered in the D2 receptor-deficient mice (data not shown), suggesting the genetic deletion did not change DA
receptor staining for this closely related receptor protein. No
staining was visible in the control experiments in
D2 WT mice in agreement with published results
using this antibody (McVittie et al. 1991), indicating
specificity for the D2 reagent in mouse tissue.
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D4 receptor protein staining in striatum also was
examined in four pairs of D4 receptor-deficient
and WT mice. In general, there was a reduced immunohistochemical
reaction in the striata of WT mice stained for D4
protein compared with WT striata stained for D2
protein as demonstrated using these antibodies in the rat (compare Fig.
1, top left and bottom left) (Ariano et
al. 1997; McVittie et al. 1991
).
D4 receptor expression was detected within medium-sized neurons and the surrounding neuropil within the striatum of the WT (Fig. 1, bottom left). This staining
pattern was lost in the D4 receptor-deficient
animals (Fig. 1, bottom middle). The background in the
D4 receptor-deficient section represents nonspecific staining. When the primary antibody was omitted, the staining looked very similar to the D4 staining
in the receptor-deficient mutant (Fig. 1, bottom right).
Immunohistochemical staining for D2 and
D3 DA receptors was unchanged in the
D4 receptor-deficient mutants (data not shown).
Membrane properties
Intracellular recordings with sharp electrodes in current clamp were obtained from 55 striatal neurons from D2 receptor-deficient, 35 striatal neurons from littermate WT control mice, 40 striatal neurons from D4 receptor-deficient, and 38 striatal neurons from littermate WT controls. All but two striatal neurons recovered and stained with biocytin were medium-sized spiny neurons. These were large aspiny neurons and electrophysiological data from these two neurons were omitted.
In general, passive and active membrane properties measured from
current-clamp recordings with sharp electrodes were similar in both
D2 and D4
receptor-deficient and their respective WT controls (Fig.
2A). In
D2 mice, RMPs for neurons obtained from WT and
receptor-deficient were 80.2 ± 1.2 versus
79.1 ± 0.9 (SE) mV, respectively; action potential amplitudes were
77.8 ± 2.0 versus 75.5 ± 1.9 mV; half-amplitude durations
for action potentials were 1.6 ± 0.1 versus 1.5 ± 0.1 ms; rheobases were 0.9 ± 0.1 versus 1.2 ± 0.1 nA; input
resistances were 36.4 ± 2.9 versus 29.6 ± 2.9 M
.
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Similarly in D4 animals RMPs were 73.8 ± 3.1 versus
78.8 ± 1.9 mV; action potential amplitudes were
76.1 ± 5.3 versus 79.4 ± 2.4 mV; action potential
half-amplitude durations were 1.5 ± 0.1 versus 1.8 ± 0.1 ms; rheobases were 0.9 ± 0.1 versus 0.9 ± 0.1 nA; and input
resistances were 27.9 ± 2.3 versus 31.6 ± 3.1 M
in
neurons from WT and receptor-deficient mice, respectively.
Spontaneous membrane depolarizations
In 55% of the neurons examined from D2
receptor-deficient mutants, large-amplitude (24 ± 2.7 mV)
long-duration (26 ± 7 s) spontaneous membrane
depolarizations occurred (Fig. 2B). These events never
occurred in WT mice (P = 0.003, Fisher exact test for
difference between frequencies in mutants and WT). These potentials were blocked by application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM; data not shown), a non-NMDA receptor antagonist. The
spontaneous depolarizations appeared similar to membrane
depolarizations observed in vivo intracellular recordings
(Wilson and Kawaguchi 1996). However, in contrast to the
in vivo depolarizations, these events were more sporadic and irregular
occurring on average about once per minute. They only rarely reached
the threshold for firing. These events also were specific to neurons
recorded from D2 receptor-deficient animals as
they were not observed in any of the striatal neurons recorded from
D4 receptor-deficient mice.
Spontaneous synaptic activity
During current-clamp recordings with sharp electrodes, low-amplitude (up to 10 mV) spontaneous synaptic activity is rarely observed in slices under the present recording conditions and was infrequent in WT mice. In contrast, in D2 receptor-deficient mice spontaneous activity was present (Fig. 3A, left). Spontaneous activity was also infrequent in the D4 receptor deficient mutant (Fig. 3B). To quantify these events, we counted the number of depolarizations exceeding 3 mV in amplitude for a 90-s epoch. Although there were slight increases in the mean frequency of these events in the D2 receptor-deficient mutants (29 ± 11 vs. 20 ± 7 events/90 s in D2 mutants and WTs, respectively), the difference was not statistically significant. The neurons obtained from the D4 receptor-deficient mutants displayed less frequent spontaneous synaptic events than those obtained from their WT controls (3.1 ± 1.5 vs. 34 ± 20.4 events/90 s in D4 mutants and WTs, respectively).
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4-AP-induced synaptic activity
In the presence of 4-AP and BIC, cells from both receptor-deficient and WT mice displayed marked increases in the frequency of synaptic events (Fig. 3, A and B, right). However, the D2 receptor-deficient mutants displayed significantly more events than their WT controls (P = 0.007; Figs. 3A, right, and 4A). Although the frequency of spontaneous events was increased markedly in the D4 receptor-deficient mutants, there was no difference with respect to values obtained from WT controls (Figs. 3B, right, and 4A). Spontaneous and 4-AP-induced synaptic activity was completely blocked by CNQX (10 µM, n = 5), a non-NMDA receptor antagonist in neurons from both receptor-deficient mutants and WT mice (Fig. 6B, right).
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After application of 4-AP and BIC, in addition to small-amplitude membrane depolarizations, some cells from receptor-deficient and WTs displayed rhythmic, large amplitude, long duration depolarizations (Fig. 3C, top). In D2 receptor-deficient mutants, more cells displayed this type of activity (42 vs. 15% in mutant vs. WT, respectively). It is possible that this activity was a reflection of epileptiform activity evoked in cortical pyramidal neurons by 4-AP and BIC, since we observed such activity in the cortex (Fig. 3C, bottom).
Spontaneous inward currents
Spontaneous synaptic activity is better observed using whole cell
patch-clamp recordings because the resolution (signal-to-noise ratio)
improves considerably. Whole cell patch-clamp recordings were obtained
from visualized medium-sized neurons in D2
receptor-deficient mutants (n = 6) and WT controls
(n = 5). Input resistances were not significantly
different between WT and D2 receptor-deficient mutants (197 ± 29 vs. 179 ± 50 M, respectively).
Spontaneous excitatory postsynaptic currents (EPSCs) were more frequent in D2 receptor-deficient mice than in their WT controls (Fig. 5A). To quantify these events, we counted the number of EPSCs exceeding 5 pA in amplitude for 3-5 min epochs. The average frequency of spontaneous events was significantly higher in D2 receptor-deficient mutants compared with WTs (P < 0.05; Fig. 4B). In contrast, the mean amplitudes of spontaneous events were similar (10.6 ± 0.7 pA in D2 receptor-deficient mice vs. 9.7 ± 0.6 pA in WTs). Normalized amplitude frequency histograms were also similar (Fig. 4C). Spontaneous EPSCs were blocked by CNQX (5 µM; n = 4) indicating they were mediated by activation of non-NMDA receptors (Fig. 5B).
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Effects of DA and D2 receptor agonists
The effects of D2 receptor antagonists and agonists were tested in current clamp with sharp electrodes and voltage clamp with patch electrodes in WT and D2 receptor-deficient neurons. In current clamp conditions spontaneous or 4-AP-induced synaptic activity was increased by sulpiride (10 µM), the D2 receptor antagonist (Figs. 4D and 6A) in neurons from WT mice. In contrast, in all WT neurons it was reduced by quinpirole (10-20 µM), the D2 agonist (Figs. 4D and 6B). In D2 receptor-deficient mutants, sulpiride (10 µM) had inconsistent effects from cell to cell, either increasing (n = 3) or decreasing (n = 2) frequencies of events by small amounts (Fig. 4D). Quinpirole was not tested in current-clamp conditions in D2 receptor-deficient mutants. The effects of DA, quinpirole, and sulpiride were not examined in the D4 receptor-deficient mutants because there were no differences between mutant and WT mice on basal measures of spontaneous activity.
|
In voltage-clamp conditions using patch electrodes, addition of DA (20 µM) (Fig. 5A) or the D2 agonist quinpirole (20 µM) reduced the frequency of EPSCs in WT [data for DA (n = 2) and quinpirole (n = 2) were pooled because there were no differences] (Fig. 4E). In D2 receptor-deficient mutants, both DA (20 µM; n = 2) and quinpirole (20 µM; n = 2) had little or no consistent effect on spontaneous EPSCs (2 cells increased slightly in frequency to quinpirole, 1 cell decreased, and 1 cell increased to DA; Fig. 4E).
Morphology
Medium-sized neurons filled with biocytin during intracellular
recordings with sharp electrodes (50 from D2
receptor-deficient and 22 from WT) did not show gross morphological
abnormalities (Figs. 7, A-C,
and 8, A-C).
Cross-sectional somatic areas were similar (135 ± 5 vs. 133 ± 6 µm2 in D2
receptor-deficient mutants and WTs, respectively). The circumference of
the dendritic field was estimated for cells that did not have truncated
dendrites by measuring the linear distance between dendritic endings.
These values were also similar (585 ± 22 vs. 577 ± 20 µm
in D2 receptor-deficient mutants and WTs, respectively). However, a subpopulation of cells from
D2 receptor-deficient mice displayed subtle
changes in spine densities and dendritic appearance (Figs.
7B and 8B). Spine densities and dendritic
appearance were rated by multiple observers blinded to genotype
according to a semi-quantitative three-point scale [1 = thin
dendrites, few spines (<2/10 µm); 2 = thick dendrites, moderate
spine density (3-5/10 µm); 3 = thick dendrites, high spine
density (>5 spines/10 µm)]. There was a statistically significant
increase in the proportion of neurons with thin dendrites and spine
densities <0.2/10 µm in D2 receptor-deficient
mutants (32% in mutants vs. 6% in WT mice; P < 0.01, 2 statistic). In this subset of neurons
dendrites sometimes displayed varicosities instead of spines.
Dye-coupling occurred in both WT and receptor-deficient animals. The
incidence of coupling was slightly higher in WT animals (12% in
D2 receptor-deficient compared with 19% in WT),
but the difference was not statistically significant.
|
|
Medium-sized neurons from D4 receptor-deficient mutants(n = 31) and WT controls (n = 32) were similar in somatic cross-sectional area (120 ± 6 vs. 132 ± 5 µm2 for mutant vs. WT, respectively), dendritic field circumference (593 ± 29 vs. 617 ± 33 µm for mutant vs. WT, respectively), dendritic appearance and spine density estimates (Figs. 7, D and E, and 8, D and E). The incidence of dye-coupling was also similar (19% in mutants vs. 22% in WT).
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DISCUSSION |
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The results of these experiments indicate that genetic deletion of the D2 receptor produces a series of chronic electrophysiological alterations consistent with an inhibitory role of this DA receptor subtype in the intact striatum. First, high-amplitude, long-duration spontaneous depolarizing events that were mediated by activation of glutamate receptors occurred only in neurons from D2 receptor-deficient mice. Second, in the presence of 4-AP and BIC, the frequency and amplitude of glutamate receptor-mediated spontaneous depolarizations were increased. Third, there was an increase in spontaneous glutamate receptor-mediated inward membrane currents.
In addition, acute modulation of D2 family receptors in WT controls altered spontaneous membrane depolarizations and inward currents mediated by activation of glutamate receptors in a manner consistent with an inhibitory function of such receptors. Interestingly, such acute modulation tended to produce milder effects than genetic deletion of the receptor.
Taken together these observations argue for an increase in glutamate transmission at the corticostriatal and possibly the thalamostriatal pathway in D2 receptor-deficient animals. Concomitant morphological changes, though subtle, suggest that the increased excitatory transmission in the D2 receptor-deficient mutant may be deleterious to a subpopulation of medium-sized striatal spiny neurons. These effects did not occur in D4 receptor-deficient mice, indicating specificity of function of D2 receptors in regulation of striatal glutamate transmission.
As pointed out in the preceding text, there is considerable evidence
for both pre- and postsynaptic localization of D2
receptors in striatum. Although our findings demonstrate an inhibitory
role for this receptor subtype in modulating glutamate
receptor-mediated responses, we cannot exclusively discriminate between
pre- and postsynaptic function. Increases in frequency of inward
currents without changes in average amplitude of events is typically
taken as evidence for a change in presynaptic modulation (Dudel
and Kuffler 1961; Takahashi et al. 1996
).
Previously, using a similar paradigm (4-AP and BIC), D2 receptors were
shown to presynaptically modulate glutamate receptor-mediated responses
in a subset of striatal neurons (Flores-Hernández et al.
1997
). The present data on the effects of quinpirole and
sulpiride in WT mice support these findings, demonstrating similar
acute effects of pharmacological manipulations. Furthermore increases
in synaptic activity in the D2 receptor-deficient
mutants would indicate the inhibitory modulation is absent. Although we
could not directly compare amplitudes of spontaneous depolarizations in
current-clamp experiments (because RMPs and input resistances were not
always the same), in voltage-clamp experiments, average amplitudes and
normalized amplitude frequency histograms of spontaneous inward
currents were similar in mutant and WT mice, providing at least some
supporting evidence that the missing inhibition could have been of
presynaptic origin. The demonstration of D2
receptors on at least a subpopulation of corticostriatal terminals
(Fisher et al. 1994
; Sesack et al. 1994
)
provides additional morphological support for a presynaptic modulatory
role of D2 receptors. Presynaptic inhibition of
glutamate release may involve DA's actions on voltage-gated calcium
channels at the presynaptic terminal. D2
receptors may be activated presynaptically to reduce calcium currents
involved in the release of glutamate (Bargas et al.
1998
; Lovinger et al. 1994
). Indeed,
D2 receptors have been shown to decrease calcium
currents in a number of systems (Berkowicz and Trombley
2000
; Koga and Momiyama 2000
; Miyazaki and Lacey 1998
; Shoji et al. 1999
). Thus lack of
D2 receptors signals the loss of an important
negative regulatory mechanism controlling glutamate release.
Previously we demonstrated that activation of D2 receptors attenuates
synaptic responses mediated by glutamate receptors in the striatum
(Levine et al. 1996b). At that time, we interpreted the
data to indicate that the effects of D2 receptors were primarily postsynaptic. Using paired-pulse facilitation as an index of
presynaptic effects, we demonstrated that application of DA did not
alter paired-pulse facilitation. However, in that study, quinpirole produced a nonsignificant increase in paired-pulse facilitation, suggesting that there may have been a presynaptic component to D2
activation in a subset of neurons, a conclusion similar to the one
reached by Flores-Hernández and colleagues (1997)
.
The failure to demonstrate presynaptic effects by activation of D2
receptors may depend on the experimental approach. Clear presynaptic
effects have been observed using chronic depletions of DA
(Calabresi et al. 1993). This is probably because after DA-depletion D2 receptors become supersensitive. Similarly, the present
study demonstrated a presynaptic inhibitory role of
D2 receptors on glutamate release because the
mutant animals have been chronically deprived of
D2 receptors.
Since we did not obtain evidence for increases in glutamate
receptor-mediated activity in D4
receptor-deficient mutants, we can conclude that the
D4 receptor probably does not have an inhibitory role in the striatum, at least with respect to responses mediated by
activation of glutamate receptors. The D4
receptor is more prevalent in the cortex than in the striatum
(Ariano et al. 1997). Interestingly, we have shown that
in the cortex of D4 receptor-deficient mice,
there is an increase in glutamate receptor-mediated synaptic activity
(Altemus et al. 1998
), suggesting an inhibitory role of
this DA receptor subtype in a different neural area.
4-AP has been shown to enhance synaptic transmission via multiple
mechanisms, depending on its concentration (Ruteki et al. 1987; Thesleff 1980
). In the hippocampus, it
produces epileptiform activity (Ruteki et al. 1987
). In
the striatum, the effects of 4-AP have been hypothesized to be due to
calcium-dependent glutamate release (Flores-Hernández et
al. 1997
). Application of 4-AP will also release other striatal
transmitters like GABA, acetylcholine, and dopamine. In the present
study, we used BIC to block effects of activation of
GABAA receptors and demonstrated that CNQX blocks the remaining depolarizations produced by the 4-AP. Thus it is highly
probable that the increase in frequency of depolarizations after 4-AP
and BIC was due to release of glutamate and activation of glutamate
receptors. One caveat though is whether spontaneous DA release is
altered in the D2 mutant. If less DA is released by 4-AP, then one could argue that there might be less influence by DA
on the remaining DA receptor subtypes. Although DA levels do not appear
altered in D2 receptor-deficient mutants
(Dickinson et al. 1999
), additional work will have to be
performed to examine this issue in more detail. However, the
observation that even in the absence of 4-AP spontaneous synaptic
events were significantly increased in whole cell recordings
demonstrates that glutamate release is facilitated in
D2 receptor-deficient mice. Application of 4-AP
only makes this effect more distinct.
The results of the present study may now provide an
electrophysiological basis for some of the previously reported
phenotypes displayed by D2 receptor-deficient
mice. For example, reduced regulatory function of DA has been
demonstrated in the substantia nigra of these mice (Mercuri et
al. 1997). In nucleus accumbens, D2
receptors have been shown to play an important role in prepulse inhibition (Ralph et al. 1999
). Alterations in the
expression of synaptic plasticity have also been observed in the
D2 receptor-deficient mouse (Calabresi et
al. 1997b
). In these mice, tetanic stimulation of the
corticostriatal pathway in slices induces long-term potentiation instead of long-term depression. Calabresi et al. (1997a
,b
,
2000
) have shown that such long-term potentiation occurs in the
striatum when the NMDA receptor is relieved of its
Mg2+ block. In light of the present findings, the
change in synaptic plasticity in D2
receptor-deficient mutants may be interpreted as an effect of
dysregulated glutamate release in the striatum and the concomitant
release of the NMDA receptor from its Mg2+ block.
Thus D2 receptors could play an important role in
controlling glutamate-evoked responses in the striatum and the
resulting direction of synaptic plasticity. The present findings also
support our proposed scheme of DA's actions that predicts a negative
regulation of glutamate receptor activation by D2
receptors (Cepeda and Levine 1998
; Cepeda et al.
1993
; Levine and Cepeda 1998
). Thus the absence of D2 receptors in the mutant mice should favor
the enhancement of excitatory synaptic activity.
Presynaptic regulation of glutamate release by DA acting on
D2 receptors may also help understand the low
levels of spontaneous activity in the striatum. Although intrinsic
potassium currents undoubtedly help keep medium-sized spiny neurons
hyperpolarized (Calabresi et al. 1987b), the role of DA
acting on D2 receptors should not be neglected.
DA released tonically could activate D2 receptors
located on corticostriatal terminals (possibly in a paracrine fashion)
to prevent excessive release of glutamate and keep the postsynaptic
membrane hyperpolarized (down-state). When highly synchronized inputs
occur, the membrane would depolarize (up-state) (see Wilson and
Kawaguchi 1996
). As we have shown, under these circumstances,
NMDA receptor blockade is relieved, and now DA, acting on D1 receptors,
will facilitate glutamate responses (Cepeda and Levine
1998
; Cepeda et al. 1993
).
The presynaptic regulation of glutamate release by
D2 receptors is better appreciated after
unilateral lesions of the substantia nigra that deplete the striatum of
DA. One of the chronic effects of such lesions is to increase
spontaneous synaptic activity and cell firing in striatal neurons
(Cepeda et al. 1989; Galarraga et al.
1987
; Schultz 1982
). This activity can be
modulated by D2 receptor agonists, possibly via presynaptic mechanisms
(Calabresi et al. 1993
).
If increased release of glutamate occurs in D2
receptor-deficient mice as we propose, structural alterations in
striatal neurons over long time periods could be expected due to
increased exposure to glutamate. The present experiments indicate that
a subpopulation of medium-sized spiny neurons labeled with biocytin
have reduced dendritic spines. These findings are consistent with a
degenerative process. In support of this interpretation, we have shown
previously that striatal cell swelling, the first step in the
excitotoxic cascade induced by exposure to NMDA or kainate, is reduced
by quinpirole in a dose-dependent fashion (Cepeda et al.
1998b).
Although the majority of evidence favors D2
receptor-mediated inhibition of corticostriatal glutamate-evoked
activity, there is another interpretation that should be considered.
Previously it had been shown that glutamate and DA are potentially
co-localized in nigrostriatal neurons (Sulzer et al.
1998). Therefore it is a possibility that DA, acting via D2
autoreceptors, could inhibit glutamate release from endings of
nigrostriatal neurons. If DA and glutamate are also colocalized in
nigrostriatal neurons in the mouse, then the deletion of inhibitory
D2 autoreceptors may predispose DA-containing
neurons to release increased quantities of glutamate and DA. As pointed
out in the preceding text, recent evidence has been provided to the
contrary. DA levels in the striatum of D2
receptor-deficient mice and their WT controls are not different (Dickinson et al. 1999
).
Receptor-deficient mouse mutants offer many advantages for the study of
the molecular and cellular mechanisms underlying receptor function.
They are especially valuable when pharmacological tools are either
unavailable or they lack the specificity to discriminate among closely
related receptor subtypes. However, as with any technique, there are
limitations. One problem with such mice has to do with the
developmental compensation that occurs in animals lacking a particular
receptor subtype (Holsboer 1997). Consequently the
effects observed in the receptor-deficient animals could be due to
changes in other remaining receptors as well as the deleted receptor.
There is no way to completely control for this possibility with the
animals currently available, but it should be minimized in future
conditional D2 receptor-deficient mutant mice
(Crawley et al. 1997
; Pich and Epping-Jordan
1998
). One approach that has been used to address the issue of
compensation at the molecular level has been to examine mice for
significant changes in the expression of the remaining DA receptor
subtypes (Saiardi et al. 1998
) as well as other relevant
proteins (Murer et al. 2000
). Another caveat associated
with mutants concerns the genetic background of the strain. Behavioral
studies using one D2 receptor-deficient mutant
suggested that this mouse was a potential model of Parkinson's disease
(Baik et al. 1995
). However, behavioral studies using the strain examined in the present experiments showed mild motor impairments in D2 mutants (Kelly et al.
1998
). Recent studies by other investigators suggest that there
is little difference in the unconditioned locomotor behaviors between
the two strains of D2 receptor-deficient mutant
mice (Boulay et al. 2000
; Clifford et al.
2000
). Moreover, the two WT background strains used in the
present studies were similar to each other in every quantified electrophysiological parameter despite large differences in genetic background. Additional experiments will need to be performed to determine precisely how strain differences may affect
electrophysiological analyses.
In conclusion, two main functions can be envisaged for
D2 receptors. First, by inhibiting glutamate
responses, they could act as a filtering device to select synaptic
inputs and increase the signal-to-noise ratio (Cepeda et al.
1993; Flores-Hernández et al. 1997
). This
function emphasizes acute effects of DA at D2
receptors. Second, D2 receptors could function as
a gatekeeper and protect striatal neurons from excessive glutamate
stimulation (Cepeda and Levine 1998
; Godukhin et
al. 1984
). Such a function would emphasize more chronic,
long-term effects of DA release and provide evidence for a paracrine
role of DA.
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ACKNOWLEDGMENTS |
---|
The authors acknowledge D. Crandall and C. Gray for the preparation of the illustrations.
This work was supported by National Institutes of Health Grants NS-33538 (M. S. Levine), NS-35649 (M. S. Levine), and DA-12062 (D. K. Grandy), by the National Alliance for Research on Schizophrenia and Depression (M. S. Levine), and by Office of Naval Research Grant N00149810436 (M. S. Levine).
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FOOTNOTES |
---|
Address for reprint requests: M. S. Levine (E-mail: mlevine{at}mednet.ucla.edu).
* C. Cepeda, R. S. Hurst, and K. L. Altemus contributed equally to this study.
Received 12 September 2000; accepted in final form 27 October 2000.
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REFERENCES |
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