1Dipartimento di Neuroscienze, Clinica Neurologica, Università di "Tor Vergata"; 2Consiglio Nazionale delle Ricerche, Istituto di Medicina Sperimentale; 3IRCCS Ospedale S. Lucia, 00133 Rome; and 4Il Clinica Neurologica, Università "La Sapienze," 00185 Rome, Italy
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ABSTRACT |
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Centonze, Diego, Paolo Gubellini, Barbara Picconi, Paolo Calabresi, Patrizia Giacomini, and Giorgio Bernardi. Unilateral Dopamine Denervation Blocks Corticostriatal LTP. J. Neurophysiol. 82: 3575-3579, 1999. The nigrostriatal dopaminergic projection is crucial for the striatal processing of motor information received from the cortex. Lesion of this pathway in rats causes locomotor alterations that resemble some of the symptoms of Parkinson's disease and significantly alters the excitatory transmission in the striatum. We performed in vitro electrophysiological recordings to study the effects of unilateral striatal dopamine (DA) denervation obtained by omolateral nigral injection of 6-hydroxydopamine (6-OHDA) in the formation of corticostriatal long-term potentiation (LTP). Unilateral nigral lesion did not affect the intrinsic membrane properties of striatal spiny neurons. In fact, these cells showed similar pattern of firing discharge and current-voltage relationship in denervated striata and in naive controlateral striata. Moreover, excitatory postsynaptic potentials (EPSPs) evoked by stimulating corticostriatal fibers and recorded from DA-denervated slices showed a pharmacology similar to that observed in slices obtained from controlateral intact striata. Conversely, in magnesium-free medium, high-frequency stimulation (HFS) of corticostriatal fibers produced LTP in slices from nondenervated striata but not in slices from 6-OHDA-denervated rats. After denervation, in fact, no significant changes in the amplitude of extra- and intracellular synaptic potentials were recorded after the conditioning HFS. The absence of corticostriatal LTP in DA-denervated striata might represent the cellular substrate for some of the movement disorders observed in Parkinson's disease.
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INTRODUCTION |
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The nigrostriatal dopaminergic projection plays a
crucial role in the physiological activity of basal ganglia. Loss of
dopamine (DA)-containing neurons of this pathway, in fact, is the main pathological characteristic of Parkinson's disease (PD). The striatum also receives extensive glutamatergic projections from the cerebral cortex and thalamus (Calabresi et al. 1996b;
Divac et al. 1977
; Lapper and Bolam
1992
), and a functional link between DA and glutamate has been
proposed on the basis of behavioral, biochemical, and physiological
studies (Graybiel 1990
; Groves 1983
;
Koetter 1994
; Starr 1995
). Spiny neurons,
accounting for the large majority of striatal cell population,
represent the main synaptic target of both DAergic input arising from
substantia nigra and glutamatergic projections arising from the cortex
and thalamus (Ariano et al. 1997
; Bouyer et al.
1984
; Freund et al. 1984
; Graybiel
1990
; Groves 1983
; Koetter 1994
;
Seasack et al. 1994
; Smith and Bolam
1990
).
Two different forms of long-term change of the efficacy of
corticostriatal synaptic transmission have been described, long-term depression (LTD) and long-term potentiation (LTP) (Calabresi et al. 1992c,d
, 1994
, 1996b
;
Lovinger et al. 1993
; Walsh 1993
).
Although in both forms of synaptic plasticity the interplay between
glutamate and DA receptors has been described (Calabresi et al.
1992c
, 1997b
; Choi and Lovinger
1997
; Lovinger et al. 1993
), the effects of DA-deafferentation has been investigated on corticostriatal LTD (Calabresi et al. 1992c
) but not LTP. Morphological
studies showed that DA-denervation causes major structural changes in
the striatum that might profoundly interfere with the generation of
this form of synaptic plasticity. In particular, after
6-hydroxydopamine (6-OHDA) denervation, a frequently used rat model of
human PD, dendritic spines of striatal neurons are numerically reduced
and present abnormal size and shape (Ingham et al. 1989
;
Nitsch and Riesenberg 1995
). Similar data were also
demonstrated in autopsy brains of PD patients (McNeill et al.
1988
). Noticeably, dendritic spines of striatal neurons have
been proposed to constitute the anatomic locus of the interaction
between glutamate and DA and also the site of the expression of
corticostriatal synaptic plasticity (Calabresi et al.
1996a
, 1997a
). Thus, the remodeling of these neuronal structures might cause an impairment of corticostriatal LTP.
Accordingly, it has been shown that dendritic spines constitute the
locus of long-term synaptic modifications associated with functional
plasticity in other CNS areas (Desmond and Levy 1990
).
In the present work we have investigated the effects of 6-OHDA-induced nigrostriatal lesion in the formation of corticostriatal LTP to study the role of DAergic pathway integrity in this form of synaptic plasticity.
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METHODS |
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Adult male Wistar rats (150-250 g, n = 32) were
used for all the experiments. The preparation and maintenance of
coronal slices have been described previously (Calabresi et al.
1990, 1994
). Briefly, corticostriatal coronal
slices (200-300 µm) were prepared from tissue blocks of the brain
with the use of a vibratome. A single slice was transferred to a
recording chamber and submerged in a continuously flowing Krebs
solution (35°C, 2-3 ml/min) gassed with 95%
O2-5% CO2. The composition
of the control solution was (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 glucose, and 25 NaHCO3.
Intracellular recording electrodes were filled with 2 M KCl (30-60
M), whereas extracellular electrodes were filled with 2 M NaCl
(5-10 M
). Intracellular and extracellular potentials were recorded
with the use of an Axoclamp 2A amplifier, displayed on an oscilloscope,
and stored in a digital system. For synaptic stimulation, bipolar
electrodes were used. These stimulating electrodes were located either
in the cortical areas close to the recording electrode or in the white
matter between the cortex and the striatum to activate corticostriatal
fibers. During tetanic stimulation the intensity was increased to
levels producing the maximal amplitude of the field potential or an
action potential on the excitatory postsynaptic potential (EPSP;
approximately twice the test intensity). The field potential amplitude
was defined as the average of the amplitude from the peak of the early
positivity to the peak negativity, and the amplitude from the peak
negativity to the peak late positivity. Quantitative data on
posttetanic modifications are expressed as percentage of the controls,
the latter representing the mean of responses recorded during a stable
period (15-30 min) before tetanic stimulation. Values given in the
text and in the figures are means ± SE of changes in the
respective cell populations. Student's t-test (for paired
and unpaired observations) was used to compare the means. Drugs were
applied by dissolving them to the desired final concentration in the
saline and by switching the perfusion from control saline to
drug-containing saline. Drug solutions entered the recording chamber
within 40 s after a three-way tap had been turned on.
DL-2-Amino-5-phosphovaleric acid (APV) and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) were from Tocris-Cookson.
To obtain unilateral nigrostriatal lesions, rats were injected with
6-OHDA (8 µg/4 µl of saline containing 0.1% ascorbic acid) via a
Hamilton syringe through a cannula inserted just rostral to the
substantia nigra under stereotaxic coordinates (Paxinos and
Watson 1986): A, 3.7 mm anterior to the interaural line; V, 2.2 mm dorsal to the interaural line; L, 2.2 mm from the midline. Twenty
days later, the rats were tested with 0.5 mg/kg sc apomorphine, and the
controlateral turns were recorded with automatic rotometers for 3 h. Only those rats consistently making at least 200 controlateral turns
were used for the electrophysiological studies. After brain dissection,
we confirmed that the nigrostriatal pathway was lesioned. This was
established by noting a >95% loss of DA neurons in the substantia
nigra compacta and the almost complete absence of DA terminals in the
striatum. This was detected by an immunoperoxidase technique, which
utilized a monoclonal antibody for tyrosine hydroxylase. Most of the
experiments were performed from rats killed 3-4 mo after the
unilateral DA-denervation.
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RESULTS |
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Electrophysiological experiments were conducted on
corticostriatal slices obtained from 6-OHDA-denervated striata and
controlateral naive striata. Intracellular recordings showed that
intrinsic membrane properties of striatal neurons were similar in the
two groups and closely resembled the electrical activity described previously for both naive (Calabresi et al. 1990,
1996b
; Cepeda et al. 1994
; Jiang
and North 1991
; Kita et al. 1984
) and
DA-denervated rat striatal neurons (Calabresi et al.
1993
). The resting membrane potential was
86 ± 4 (SE)
mV in unlesioned striata (n = 37) and
85 ± 4 in
DA-lesioned striata (n = 40; P > 0.05). In both groups, neurons were silent at rest, and the injection
of positive current (0.6-1.0 nA; Fig.
1A and B) through
the recording pipette induced a tonic firing discharge. Voltage-clamped
neurons, at membrane potentials close to the resting level (
85 mV),
from DA-denervated and naive striata displayed similar responses to
voltage steps (0.5-3 s duration) of increasing amplitude shifting the
membrane in depolarizing and hyperpolarizing directions (from
120 to
40 mV; n = 10 for each experimental group; Fig.
1B). Membrane rectification was present in both groups
(Calabresi et al. 1990
, 1996b
;
Jiang and North 1991
; Kita et al. 1984
).
Also synaptic potentials evoked by stimulation of corticostriatal
glutamatergic fibers were similar in naive and DA-denervated slices. In
fact, in control medium extracellular field potentials (FPs) and
intracellulary recorded EPSPs were not affected by the
N-methyl-D-aspartate (NMDA) glutamate receptor antagonist APV (30 µM, n = 8 for each
experimental group) but were almost completely abolished by
coadministration of 30 µM APV plus 10 µM CNQX, an
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate
receptor antagonist, in both groups of animals (n = 8 for each experimental group; Fig. 1, Ca and
Cb). In magnesium-free solution, a procedure that
deinactivates NMDA receptors, both FPs and EPSPs increased, unmasking
an APV-sensitive component that was similar in naive and DA-denervated
striata. In fact, under this condition the pharmacological application
of 30 µM APV produced a significant reduction of the EPSP amplitude
and duration. The amplitude of this pharmacological effect was similar in both naive and DA-denervated striata (n = 8 for
each experimental condition). The coadministration of APV and CNQX
fully blocked corticostriatal synaptic transmission in both the
experimental groups (n = 8; Fig. 1,
Cc and Cd).
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As previously reported (Calabresi et al. 1992d), in the
absence of magnesium ions from the external medium, high-frequency stimulation (HFS; 3 trains, 3 s duration, 100-Hz frequency, 20-s interval) of corticostriatal fibers produced, in the intact striata, an
APV-sensitive LTP of extracellularly recorded FPs
(n = 15, P < 0.001; Fig.
2, Aa and
Ab). Similar data were obtained by using intracellular
recordings from intact striata (n = 15, P < 0.001; Fig. 2, Ba and
Bb). Conversely, in DA-denervated slices the same stimulation failed to induce LTP. In these slices, in fact, no significant changes in the amplitude of FPs (n = 15, P > 0.05; Fig. 2, Ac and
Ad) and EPSPs (n = 15; Fig. 2,
Bc and Bd) were observed after the
conditioning HFS (P > 0.05).
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DISCUSSION |
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In the present study we provide evidence for the absence of LTP in the striatum of DA-denervated striata. Noticeably, the absence of post-tetanic LTP is coupled neither to changes of intrinsic membrane properties of the recorded striatal neurons (resting membrane potential and current-voltage relationship) nor to alterations in the pharmacology of the corticostriatal synaptic potentials evoked by single cortical activation.
A critical role of DA in the long-term regulation of the efficacy of
excitatory transmission in the striatum has been demonstrated by
previous electrophysiological findings. Coactivation of D1-like and
D2-like receptors, in fact, is required for the induction of
corticostriatal LTD (Calabresi et al. 1992b,c
;
Choi and Lovinger 1997
), whereas D2-like receptors
modulate corticostriatal LTP (Calabresi et al. 1997b
).
In mice lacking D2 DA receptors, the amplitude of HFS-induced LTP is
higher than in control condition, closely resembling the values
obtained following the acute blockade of D2-like receptors in slices
prepared from control animals (Calabresi et al. 1997
).
The latter observation strengthens the idea that the integrity of
DAergic pathway, and not simply the expression of DA receptors, allows
the induction of LTP. The permanent disruption of D2 DA receptor
encoding gene, in fact, is not sufficient to mimic the chronic
DA-denervation condition.
The integrity of DAergic input from the substantia nigra to the
striatum is crucial for the physiological activity of basal ganglia and
various pre- and postsynaptic short-term effects of DA have been
described in striatal neurons (Calabresi et al. 1987, 1992a
, 1993
; Cepeda et al.
1998
; Surmeier et al. 1992
,
1995
). Accordingly, the loss of DAergic modulation of
striatal synaptic plasticity has been proposed to represent the
cellular substrate for parkinsonian symptoms (Calabresi et al.
1996
; 1997b
) and abnormal synaptic plasticity,
in the absence of changes of other electrophysiological parameters, has
been found in the striatum of mice lacking D2 receptors
(Calabresi et al. 1997b
), which present a
parkinsonian-like phenotype (Baik et al. 1995
). However,
the lesion of nigrostriatal DAergic pathway by 6-OHDA injection is the
most widely used animal model of PD and causes major morphological and
functional changes in the striatum that closely resemble alterations
described in parkinsonian patients. Both an increased turnover of DA in
the surviving DAergic neurons and a DA receptor supersensitivity have been demonstrated in the striatum in 6-OHDA-treated rats and also in
PD (Calabresi et al. 1993
; Hornykiewicz
1993
; Zigmond et al. 1990
). In addition, an
increased concentration and release of glutamate from corticostriatal
terminals has been reported in the striatum following DA-denervation
(Lindefors and Ungerstedt 1990
), and morphological and
electrophysiological findings strongly support this idea. In
6-OHDA-treated rats (Ingham et al. 1993
) and also in PD
(Anglade et al. 1996
), in fact, a significant increase in the length of the postsynaptic densities of corticostriatal synapses
has been found, suggesting a hyperactivity of these synapses. Furthermore, in vitro electrophysiological recordings showed after nigral lesion a prominent enhancement of spontaneous depolarizing postsynaptic potentials in rat striatal neurons that are blocked by the
glutamate receptor antagonist CNQX (Calabresi et al.
1993
). Thus the increased release of glutamate in the striatum
following DA-deafferentation might represent a plastic compensatory
adaptation to the observed loss of long-term facilitation of
corticostriatal glutamatergic transmission exerted by DA. Accordingly,
in both 6-OHDA-treated animals and PD, motor symptoms become apparent when a great proportion of nigrostriatal neurons is lost.
In conclusion, two different hypotheses might explain the absence of LTP after DA-denervation. First, the loss of this form of synaptic plasticity might be due to the absence of endogenous DA after unilateral 6-OHDA-induced nigral lesion. Alternatively, it is also possible that the profound morphological adaptive synaptic changes observed in the striatum following chronic denervation play a role in the disruption of this form of synaptic plasticity. We are planning future experiments using exogenous DA and DAergic agonists in DA-denervated slices to investigate whether the activation of DA receptors might restore post-tetanic LTP. These future experiments might provide conclusive data concerning this issue.
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ACKNOWLEDGMENTS |
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We thank M. Tolu for the technical assistance.
This study was supported by a grant from BIOMED Project to P. Calabresi (No. BMH4-97-2215), by a grant from TELETHON Project to P. Calabresi (No. E0.729), and by a Ministero dell'Università e della Ricerca Scientifica e Tecnologica grant (Cofinanziamento) to P. Calabresi.
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FOOTNOTES |
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Address for reprint requests: P. Calabresi, Clinica Neurologica, Dipartimento di Neuroscienze, Università di Roma "Tor Vergata," via di Tor Vergata 135, 00133 Rome, Italy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 May 1999; accepted in final form 22 July 1999.
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REFERENCES |
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