Center for Molecular and Behavioral Neuroscience, Program in Cellular and Molecular Biodynamics, Rutgers, The State University of New Jersey, Newark, New Jersey 07102
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ABSTRACT |
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Iribe, Yuji,
Kevin Moore,
Kevin C. H. Pang, and
James M. Tepper.
Subthalamic Stimulation-Induced Synaptic Responses in Substantia
Nigra Pars Compacta Dopaminergic Neurons In Vitro.
J. Neurophysiol. 82: 925-933, 1999.
The subthalamic
nucleus (STN) is one of the principal sources of excitatory
glutamatergic input to dopaminergic neurons of the substantia nigra,
yet stimulation of the STN produces both excitatory and inhibitory
effects on nigral dopaminergic neurons recorded extracellularly in
vivo. The present experiments were designed to determine the sources of
the excitatory and inhibitory effects. Synaptic potentials were
recorded intracellularly from substantia nigra pars compacta
dopaminergic neurons in parasagittal slices in response to stimulation
of the STN. Synaptic potentials were analyzed for onset latency,
amplitude, duration, and reversal potential in the presence and absence
of GABA and glutamate receptor antagonists. STN-evoked depolarizing
synaptic responses in dopaminergic neurons reversed at approximately
31 mV, intermediate between the expected reversal potential for an
excitatory and an inhibitory postsynaptic potential (EPSP and IPSP).
Blockade of GABAA receptors with bicuculline caused a
positive shift in the reversal potential to near 0 mV, suggesting that
STN stimulation evoked a near simultaneous EPSP and IPSP. Both synaptic
responses were blocked by application of the glutamate receptor
antagonist, 6-cyano-7-nitroquinoxalene-2,3-dione. The confounding
influence of inhibitory fibers of passage from globus pallidus and/or
striatum by STN stimulation was eliminated by unilaterally transecting
striatonigral and pallidonigral fibers 3 days before recording. The
reversal potential of STN-evoked synaptic responses in dopaminergic
neurons in slices from transected animals was approximately
30 mV.
Bath application of bicuculline shifted the reversal potential to ~5
mV as it did in intact animals, suggesting that the source of the IPSP
was within substantia nigra. These data indicate that electrical
stimulation of the STN elicits a mixed EPSP-IPSP in nigral dopaminergic
neurons due to the coactivation of an excitatory monosynaptic and an
inhibitory polysynaptic connection between the STN and the dopaminergic
neurons of substantia nigra pars compacta. The EPSP arises from a
direct monosynaptic excitatory glutamatergic input from the STN. The
IPSP arises polysynaptically, most likely through STN-evoked excitation
of GABAergic neurons in substantia nigra pars reticulata, which
produces feed-forward GABAA-mediated inhibition of
dopaminergic neurons through inhibitory intranigral axon collaterals.
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INTRODUCTION |
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In vivo dopaminergic neurons fire spontaneously at
relatively low rates in a regular or pacemaker-like mode, a random mode or in an irregular pattern punctuated with slow bursts typically comprising two to six action potentials (Bunney et al.
1973; Grace and Bunney 1984a
,b
; Tepper et
al. 1995
; Wilson et al. 1977
). In vitro, under
control conditions, dopaminergic neurons in adults fire only in the
pacemaker-like mode (Grace 1987
). This difference suggests that afferent input to nigral dopaminergic neurons plays an
important role in regulating their neuronal activity.
The burst firing pattern and its afferent control have generated
considerable interest, in part because burst firing may alter the
dynamics of extracellular dopamine levels in a nonlinear fashion (Gonon 1988).
N-methyl-D-aspartate (NMDA)-receptor activation has been proposed to contribute to this bursting activity
(Chergui et al. 1994
; Johnson et al.
1992
; Overton and Clark 1992
; Seutin et
al. 1994
). The three principal glutamatergic inputs to
substantia nigra arise from the frontal cortex, STN, and the
pedunculopontine nucleus (Jackson and Crossman 1983
;
Kanazawa et al. 1976
; Kita and Kitai
1987
; Naito and Kita 1994
; Van Der Kooy
and Hattori 1980
).
Although the subthalamic efferents are believed to be predominantly or
exclusively glutamatergic and thus excitatory and form asymmetric
contacts with nigral and entopeduncular neurons (Bevan et al.
1994; Chang et al. 1984
; Hammond et al.
1978
; Shink et al. 1996
), reports of the effects
of STN stimulation on the activity of substantia nigra dopaminergic
neurons in vivo are somewhat contradictory. In the earliest report,
electrical stimulation of the subthalamic nucleus was found to be
excitatory to dopaminergic and nondopaminergic nigral neurons
(Hammond et al. 1978
). In a subsequent study that used
local infusions of bicuculline to stimulate the subthalamic nucleus
pharmacologically, approximately equal numbers of excitatory and
inhibitory responses were found among dopaminergic neurons, although
almost all of the nondopaminergic neurons in pars reticulata were
excited (Robledo and Féger 1990
). Similarly, when
subthalamic neurons were inhibited by local infusions of muscimol, less
than one-fourth of the dopaminergic neurons recorded showed the
expected decrease in firing rate, whereas approximately half showed
excitation and the remainder a biphasic or no effect. In contrast, 8 of
10 nondopaminergic pars reticulata neurons were inhibited. More
recently, biphasic effects of electrical stimulation of subthalamic
nucleus on electrophysiologically identified dopaminergic neurons were
reported, although the initial effect on all dopaminergic neurons as
well as on many nondopaminergic neurons was inhibition (Smith
and Grace 1992
).
The inhibitory responses in dopaminergic neurons are likely an indirect
effect, resulting from STN stimulation-induced activation of inhibitory
neurons. Early extracellular recording studies showed a reciprocal
relationship between the activity of dopaminergic neurons in pars
compacta and nondopaminergic neurons in pars reticulata, suggesting
that GABAergic neurons in the substantia nigra pars reticulata may
modulate the activity of dopaminergic neurons in pars compacta
(Grace and Bunney 1979; Grace et al.
1980
). Direct evidence for a functional inhibitory connection
between pars reticulata GABAergic neurons and pars compacta
dopaminergic neurons was provided by the demonstrations that electrical
stimulation of the pars reticulata produced GABAergic inhibitory
postsynaptic potentials (IPSPs) in pars compacta dopaminergic neurons
in vitro (Hajós and Greenfield 1994
), selective
activation of pars reticulata GABAergic projection neurons inhibits
pars compacta dopaminergic neurons in vivo through activation of
GABAA receptors (Tepper et al.
1995
), and intracellularly labeled nigrothalamic projection neurons make synapses onto dopaminergic pars compacta neurons (Damlama 1994
). These data suggest that inhibitory
responses of pars compacta dopaminergic neurons to STN stimulation
could arise disynaptically through activation of pars reticulata
GABAergic neurons.
The present study was designed to determine the source of inhibition in substantia nigra pars compacta dopaminergic neurons after electrical stimulation of the STN by recording subthalamic stimulation-induced synaptic responses in electrophysiologically identified pars compacta dopaminergic neurons in an in vitro preparation preserving the connections between the subthalamic nucleus and the substantia nigra.
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METHODS |
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Slice preparation
Young adult male Sprague-Dawley rats (4-8 wk of age, 100-200 g, Zivic-Miller) were used. Animal care and surgical procedures were performed in accordance with the guidelines of the U.S. Public Health Service manual, "Guide for the Care and Use of Laboratory Animals," and were approved by the Rutgers University Institutional Review Board. Animals were anesthetized with ketamine (100 mg/kg ip) and perfused with ice-cold artificial cerebrospinal fluid (ACSF), which contained (in mM) 125 NaCl, 3.0 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1.5 MgCl2, 2.5 CaCl2, and 10 glucose. The brain was removed quickly and trimmed to a block containing the midbrain. Parasagittal sections (350-400 µm) were cut with a Campden Instruments Vibroslice and transferred to a holding tank constantly perfused with oxygenated ACSF at 32°C for 2-6 h before being transferred to the recording chamber. Oxygenated ACSF was supplied to both holding and recording chambers (0.3 ml) at a rate of 2-4 ml/min.
Hemitransections
Hemitransections were made 3 days before recording. Three days
was selected as a survival time long enough to ensure degeneration of
striatonigral and striatopallidal fibers (Nitsch and Riesenberg 1988) but short enough to minimize anterograde degenerative
changes in nigral neurons that occur after deafferentation (Saji
and Reis 1987
). Rats were anesthetized with ketamine (80 mg/kg)
and xylazine (12 mg/kg) intraperitoneally and mounted in a stereotaxic
frame. Using aseptic surgical techniques a narrow burr hole ~3 mm
long was drilled from coordinates L 0.9 to L 3.9 mm overlying the
globus pallidus. A knife blade was lowered to 9.0 mm from the cortical surface and moved mediolaterally two or three times to produce a
unilateral transection anterior to STN. Successful lesions were accompanied by ipsilateral rotations in the animal that were usually noticeable as soon as the animal awoke and persisted until the animal
was killed. All lesions were evaluated under a stereomicroscope before
recording. Slices from brains in which the lesion did not completely
transect the midbrain and internal capsule throughout their
dorsoventral extent just anterior to the subthalamic nucleus were
excluded from study.
Intracellular recording
Intracellular recording electrodes were made from 1.2-mm OD
capillary tubing on a Sutter Instruments P-97 horizontal pipette puller. They were filled with 1 M potassium acetate containing 1%
biocytin and possessed in vitro impedances of 80-120 M. Electrode signals were amplified by a Neurodata IR183 preamplifier and displayed on a Tektronix 5111A oscilloscope. Traces were digitized on a Nicolet
4094C digital oscilloscope and transferred to a Macintosh computer for
off-line analysis with custom designed software.
Stimulation
Stimulating electrodes were bipolar stainless wires (100 µm diam) insulated with enamel except for the tips (California Fine Wire). These electrodes were placed on the surface of the slice at the level of the STN near the caudal pole of the nucleus. Stimuli were generated by a Winston A-65 timer and SC-100 constant current stimulus isolation unit and consisted of single square wave pulses (0.1-0.5 ms in duration, 0.1-2.0 mA) delivered at 1 Hz. To test for N-methyl-D-aspartate (NMDA)-receptor-mediated events, in a few cases brief high-frequency trains (interpulse interval, 3 ms; pulse duration, 100 µs) of pulses (10 ms in duration, 0.1-2.0 mA) were applied at 0.25 Hz.
Reversal potential data analysis
Reversal potentials were calculated by recording the synaptic
response to STN stimulation at various membrane potentials produced by
injection of a steady state current or 300-ms pulses Synaptic responses
were measured by subtracting baseline, as measured several milliseconds
before simulation, from the response amplitude, measured at the peak of
the synaptic response. Reversal potentials were extrapolated from a
linear regression of PSP amplitude versus membrane potential over a
restricted range of the I-V curve, typically between 50
and
100 mV. The PSP amplitude data were well fit with a linear
regression over this range as indicated by the values for the
regression coefficients under control conditions which ranged from
0.864 to 0.993 (0.961 ± 0.015; mean ± SE).
Synaptic potentials were examined under control conditions and after bath application of 50 µM bicuculline, a GABAA-receptor antagonist, 50 µM 2-OH saclofen, a GABAB-receptor antagonist, or 20 µM 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), a competitive non-NMDA glutamate receptor antagonist. To investigate the participation of NMDA receptors in the PSP, in some cases brief trains of pulses were used and 10 µM MK-801 was applied. Reversal potentials were measured after 10 min of drug application. Drug applications were followed by 1-h washout period.
The effects of drugs on the reversal potential were tested for significance using a two-tailed Student's t-test. All numerical data are expressed as mean ± SE.
Drugs
Drugs were stored in frozen aliquots that were dissolved in the
perfusing solution just prior to each experiment in the concentration indicated. All drugs were applied in the bath. Complete exchange of the
bath solutions occurred within 2 min. ()-Bicuculline methchloride (bicuculline), 2-hydroxysaclofen, CNQX, and (+)-MK-801 hydrogen maleate
(MK-801) were purchased from Research Biochemicals International.
Histology
At the end of some experiments, neurons were labeled
intracellularly with biocytin by applying depolarizing pulses (1 nA, 300-ms pulses at a 50% duty cycle) for 10 min. Slices were stored in
4% paraformaldehyde in 0.15 M phosphate buffer, pH 7.4, for 2 h
at room temperature and rinsed in 0.15 M phosphate buffer overnight at
4°C. Slices were subsequently resectioned into 70-µm sections and
processed for visualization of biocytin by using a modification of the
procedure originally described by Horikawa and Armstrong
(1988) with 3,3'-diaminobenzidine (DAB) as the chromogen.
Some sections were also processed for tyrosine hydroxylase (TH)
immunocytochemistry by standard methods previously reported (Tepper et al. 1994). In these cases, biocytin-injected
neurons used nickel-enhanced DAB as the chromogen and TH immunostaining used DAB as the chromogen.
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RESULTS |
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Neuronal identification
Dopaminergic neurons were identified based on their morphology,
location within the pars compacta and electrophysiological properties
previously described from in vitro recordings (Grace 1987; Kita et al. 1986
; Lacey et al.
1987
; Nakanishi et al. 1987
; Yung et al.
1991
). Dopaminergic neurons had resting membrane potentials between -50 and -70 mV. Most neurons were spontaneously active and
fired regularly on depolarization (Fig.
1A). All cells had long
duration action potentials (>2 ms, Fig. 1B) and were
followed by pronounced afterhyperpolarizations, as shown in Fig.
1, A and C. Cells also exhibited a slowly
activating inward rectification when hyperpolarized as shown in Fig. 1,
C and D.
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Recordings were made from 75 substantia nigra pars compacta
dopaminergic neurons. Fifty of these neurons were recorded from slices
taken from intact rats and 25 from slices taken from rats hemitransected between the STN and the globus pallidus. The morphology of intracellularly labeled dopaminergic pars compacta cells has been
described previously (Grace and Onn 1989; Kita et
al. 1986
; Preston et al. 1981
; Tepper et
al. 1987
; Yung et al. 1991
). Biocytin-stained dopaminergic neurons displayed a variety of somatic shapes including fusiform, triangular and multipolar. A typical biocytin-stained dopaminergic neuron is illustrated in Fig.
2. Dopaminergic somata gave rise to
between two and five thick, smooth principal dendrites that branched
infrequently. Higher order dendrites exhibited infrequent spine-like
appendages. One population of dendrites remained largely within pars
compacta and extended for several hundred microns in a flattened disk
around the cell body. Almost all neurons extended one and more rarely
two dendrites deep into pars reticulata, perpendicular to the borders
of pars compacta. This was invariably the largest dendrite issued by
the neuron and extended for distances up to 1,200 µm, even in the
350-400 µm slices used for in vitro recording as illustrated in Fig.
2.
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The input resistance was calculated from injection of small
hyperpolarizing current pulses (-0.05 to -0.15 nA) (Fig.
1C, open circle). The mean input resistance was 156.3 ± 11.3 M (n = 15). Spike threshold was -39.9 ± 0.5 mV (n = 102 spikes from 12 cells). Resting
membrane potential was measured at the point where the membrane
potential showed an inflection after the large spike afterhyperpolarization following a spontaneous spike (see Grace and Onn
1989
) and averaged
59.4 ± 0.5 mV (n = 121 spikes from 12 cells). Most neurons were spontaneously active, with a
mean firing rate of 2.41 ± 0.19 Hz (n = 12). The
mean spike duration was 2.65 ± 0.05 ms (n = 41).
Response to STN Stimulation
Stimulation of the STN with a single pulse elicited a depolarizing postsynaptic potential (DPSP) in dopaminergic neurons. The mean stimulus current evoking the maximal amplitude DPSP was 855 ± 58 µA (n = 56). The onset latency of the DPSP was 2.71 ± 0.15 ms (n = 49) and usually remained constant at different stimulus strengths, suggesting that it was mediated monosynaptically. Under control conditions in slices from intact rats, the reversal potential for the DPSP was -31.6 ± 4.1 mV (n = 19), a value intermediate between that expected for an IPSP and EPSP, suggesting that the DPSP was comprised of a near simultaneously occurring EPSP and IPSP. These properties are illustrated for one representative dopaminergic neuron in Fig. 3.
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Effects of GABAA receptor antagonists
After application of bicuculline, a selective
GABAA receptor antagonist, the reversal potential
was shifted in the depolarizing direction to -0.8 ± 3.3 mV
(n = 16) as shown for one representative neuron in Fig.
3. The effects of the GABAB receptor antagonist, 2-hydroxysaclofen was tested in 5 neurons. 2-hydroxysaclofen failed to
shift the reversal potential in the depolarizing direction, rather
shifting it slightly but nonsignificantly in the hyperpolarizing direction to -34.8 ± 4.8 mV (n = 5). Since
GABAB IPSPs can be elicited in midbrain
dopaminergic neurons in vitro even by single pulse stimuli
(Cameron and Williams 1993), it was important to determine whether GABAB receptors might play a
role in the IPSP. The fact that bicuculline, but not 2-hydroxysaclofen
completely eliminated the IPSP component indicates that it is mediated
by GABAA receptors.
Effects of glutamate receptor antagonists
The effects of the non-NMDA receptor antagonist, CNQX (20 µM), were tested in 8 additional neurons, all of which had mixed responses in the control condition (control DPSP reversal potential = -38.1 ± 4.3 mV, n = 8). In 5 of these neurons, CNQX completely abolished the response to STN stimulation. In these cases, when CNQX was allowed to wash out, bicuculline administration shifted the reversal potential in the positive direction as in controls. These data suggest that in these cases, the IPSP component of the DPSP is polysynaptically mediated. A representative example of a polysynaptically mediated EPSP-IPSP is shown in Fig. 4.
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In the 3 remaining neurons CNQX did not abolish the DPSP but instead shifted its reversal potential to -61.1 ± 6.4 mV (n = 3), close to the expected reversal potential for a pure GABAA-mediated IPSP. After washout, subsequent administration of bicuculline shifted the DPSP reversal potential to -6.5 ± 3.1 mV (n = 3). Thus in these cases, the IPSP component of the DPSP was mediated, at least in part, monosynaptically. An example of a monosynaptically mediated EPSP-IPSP is shown in Fig. 5.
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In most cases when the DPSP was elicited by single pulse stimulation of the STN, the excitatory component could be completely blocked by the non-NMDA receptor antagonist, CNQX, as described above. However, in a few neurons, short trains of stimulation were applied to the STN. Under these conditions, a DPSP resulted that could not be completely blocked by 20 µM CNQX alone, but that could be blocked by the addition of 10 µM MK-801 (data not shown).
Effects of hemitransections
Recordings were also obtained from 25 dopaminergic neurons in
slices from 21 animals that were given complete unilateral
hemitransections between the globus pallidus and the STN 3 days prior
to recording to eliminate any confounding effects of stimulation of
descending GABAergic axons from striatum and/or globus pallidus. The
basic electrophysiological properties of dopaminergic neurons from
transected animals did not differ from those in slices from intact
rats, although the STN-evoked DPSP appeared to have a greater NMDA
component in slices from transected rats. For this reason, some of the
experiments in transected rats were conducted with MK-801 (10 µM)
present throughout. The mean input resistance was 138.8 ± 7.5 M (n = 25), the spike threshold was -39.8 ± 0.5 mV (n = 93 spikes from 11 neurons), and the resting
membrane potential was -59.6 ± 0.6 mV (n = 72 spikes from 11 neurons). The reversal potential of the STN-evoked DPSP
in control ACSF was
30.3 ± 5.0 mV (n = 9). Addition of bicuculline to the bath removed the IPSP component, leaving
a pure EPSP (reversal potential = 5.0 ± 3.0 mV,
n = 9). In 7 of these neurons, after bicuculline was
washed out for 1 h and the DPSP returned, CNQX was applied and
completely abolished the DPSP, as shown for one representative neuron
in Fig. 6A. In two additional
neurons application of 20 µM CNQX completely abolished the DPSP just
as it did following washout of bicuculline. In these two cases the
reversal potential of the DPSP under control conditions in the presence
of 10 µM MK-801 was -20.5 and -47.1 mV. Following a 1 h
washout, the DPSP returned and subsequent application of bicuculline
left a pure EPSP in both cells (reversal potential of 8.1 mV and -10.4
mV, respectively). One of these neurons is illustrated in Fig.
6B.
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DISCUSSION |
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The response to STN stimulation is a mixed EPSP-IPSP
Under control conditions STN stimulation resulted in a DPSP in
nigral dopaminergic neurons with a mean onset latency of 2.7 ms. This
latency is in good agreement with that previously reported for
excitatory responses of nigral neurons to subthalamic stimulation in
vivo (Hammond et al. 1978 1983
;
Nakanishi et al. 1987
). In most cases the onset latency
of the DPSP was invariant with changes in stimulus amplitude suggesting
that it was monosynaptic (Park 1987
). The DPSP had a
mean reversal potential around -30 mV, more depolarized than threshold
for dopaminergic neurons (-40 mV), indicating that it is effectively
an EPSP. However, -30 mV is considerably more hyperpolarized than the
expected reversal potential for glutamate mediated EPSPs in the
mammalian CNS (Puil and Benjamin 1988
). After
application of bicuculline the reversal potential was shifted in the
depolarizing direction to the expected value near 0 mV. This indicates
that the STN stimulation produced a compound synaptic response
consisting of a near simultaneous EPSP and a
GABAA-mediated IPSP.
Although it is possible that stimulation of STN produced antidromic
excitation in PPN that was conveyed orthodromically to substantia nigra
through nigral afferents from the PPN, the short latency of the
response is incompatible with the known axonal conduction times of
efferents from PPN to the STN and the substantia nigra (Scarnati
et al. 1986; Takakusaki et al. 1996
,
1997
). Furthermore, unlike PPN efferents to substantia
nigra which are both cholinergic as well as glutamatergic, the
STN-stimulation-induced excitation could be completely blocked by CNQX
(see below) whereas some effects of PPN stimulation on dopaminergic
neurons are cholinergic (Clarke et al. 1987
;
Nijima and Yoshida 1988
). Thus we conclude that the excitatory portion of the response is mediated by glutamatergic efferents from the STN.
The origin of the IPSP component
Since the efferents from the STN to substantia nigra are
exclusively glutamatergic and excitatory (Bevan et al.
1994; Chang et al. 1984
; Kita and Kitai.
1987
; Shink et al. 1996
), the inhibitory component of the DPSP almost certainly does not originate
monosynaptically from the STN. There are at least two possibilities for
the origin of the GABAA component. The STN
stimulation could activate descending GABAergic fibers from the
striatum and/or globus pallidus traveling in the fiber bundles
surrounding the STN, thereby producing a monosynaptic IPSP as suggested
by others (Nakanishi et al. 1987
; Smith and Grace
1992
). In addition, GABAergic pars reticulata projection
neurons and perhaps interneurons may exert feedforward inhibition on
pars compacta dopaminergic neurons following subthalamic stimulation by
a disynaptic (or polysynaptic) pathway (Robledo and Féger
1990
; Smith and Grace 1992
; Tepper et al.
1995
).
Both striatal and pallidal inputs to dopaminergic neurons have been
identified anatomically (Hattori et al. 1975;
Somogyi et al. 1981
; Smith and Bolam
1990
) and in vivo recordings show that striatal or pallidal
stimuli result in IPSPs and/or inhibition of firing of
electrophysiologically identified dopaminergic neurons (Grace
and Bunney 1985
; Paladini et al. 1999
;
Tepper et al. 1987
1990). In vivo studies
indicate that the IPSP produced in nigral dopaminergic neurons by
stimulation of either striatum or globus pallidus is mediated
predominantly or exclusively by GABAA receptors (Grace and Bunney 1985
; Paladini et al.
1999
).
When CNQX was administered to block the EPSP component of the response,
a pure IPSP with a reversal potential of -61.1 ± 6.4 mV remained
in 3 of 8 neurons, indicating that in these cases, the STN stimulation
did indeed activate descending inhibitory efferents as well as
excitatory subthalamic efferents. This was in fact suggested as the
most likely source of mixed EPSP and IPSP responses following
electrical stimulation of the subthalamo-nigral pathway in prior in
vitro intracellular recording studies (Nakanishi et al.
1987, 1997
) and as one of the possibilities for
the initial inhibition seen in previous in vivo extracellular
experiments (Smith and Grace 1992
). However, in 5 of 8 neurons, application of CNQX completely eliminated both the EPSP and
the IPSP indicating that in these cases, the IPSP was not caused by
stimulation of descending GABAergic afferents but rather by the
activation of an excitatory glutamatergic input to a GABAergic neuron
within the slice.
This interpretation is supported by the results obtained with slices
from rats transected anterior to the STN 3 days prior to recording to
eliminate descending GABAergic fibers from striatum and globus
pallidus. In many neurons recorded from these slices, stimulation of
the STN produced mixed EPSP-IPSPs in which the IPSP could be blocked by
either CNQX or bicuculline just as in slices from intact rats. Thus,
although subthalamic stimulation can produce a monosynaptic IPSP
through inadvertent activation of descending GABAergic nigral afferents
as well as a monosynaptic EPSP from the glutamatergic subthalamo-nigral
pathway, a polysynaptic IPSP mediated by GABAA
receptors is also evoked by subthalamic stimulation of a GABAergic
neuron somewhere in substantia nigra, most likely in pars reticulata,
as previously suggested (Nakanishi et al. 1987;
Robledo and Féger 1990
; Smith and Grace
1992
).
In an early extracellular recording study, the firing of pars compacta
dopaminergic neurons and GABAergic neurons in pars reticulata was shown
to be reciprocally related (Grace and Bunney 1979). More
recently, selective stimulation of pars reticulata GABAergic projection
neurons achieved by antidromic activation from thalamus or tectum was
shown to inhibit nigrostriatal dopaminergic neurons (Paladini et
al. 1999
; Tepper et al. 1995
), and the effects of pharmacological excitation and inhibition of globus pallidus on
nigral dopaminergic neurons were shown to be mediated indirectly through pars reticulata GABAergic neurons (Celada et al.
1999
). Intracellular labeling of pars reticulata projection
neurons in vivo shows that these neurons have extensive local
collaterals both within pars reticulata and pars compacta
(Grofova et al. 1982
) and make synapses with somata and
proximal dendrites of dopaminergic neurons in pars compacta
(Damlama 1994
). Stimulation of pars reticulata in slices
taken from animals 24-48 h after hemisection through the caudal
hypothalamus revealed IPSPs in pars compacta dopaminergic neurons
(Hajós and Greenfield 1994
). Thus, pars reticulata
GABAergic neurons, including but perhaps not restricted to
nigrothalamic and nigrotectal projection neurons, inhibit pars compacta
dopaminergic neurons via their axon collaterals both in vivo and in vitro.
Although there is clearly a monosynaptic projection from the STN to
dopaminergic neurons, there are few boutons in pars compacta. The vast
majority of subthalamic nigral efferents terminate in pars reticulata,
mostly on the dendrites of nondopaminergic neurons, but also on
dopaminergic dendrites (Damlama 1994; Kita and
Kitai 1987
). Since the pars reticulata GABAergic neurons make
functional inhibitory connections with pars compacta dopaminergic
neurons, in addition to a monosynaptic EPSP, stimulation of the STN
also leads to a disynaptic IPSP that is mediated through the local axon
collaterals of substantia nigra pars reticulata GABAergic neurons. A
similar role for pars compacta GABAergic interneurons cannot be excluded.
Effects of GABAB receptor blockade
Blockade of GABAB receptors by
2-hydroxysaclofen did not shift the reversal potential in the
depolarizing direction as did the GABAA
antagonist, bicuculline. This is consistent with other data that
demonstrate that although there is a significant postsynaptic GABAergic
tone exerted on nigral neurons through GABAA
receptors in vivo and in vitro, there does not appear to be a
significant amount of endogenous GABAB
stimulation either in vivo or in vitro (Engberg et al.
1993; Häusser and Yung 1994
;
Paladini and Tepper 1999
; Paladini et al.
1999
; Rick and Lacey 1994
).
Although it did not reach statistical significance, application of
2-hydroxysaclofen shifted the reversal potential in the opposite
direction as bicuculline, toward a more. hyperpolarized potential. This
is consistent with the ability of GABAB receptor agonists to attenuate GABAA-mediated IPSPs in
dopaminergic neurons in vitro (Häusser and Yung
1994) and of GABAB antagonists to augment
GABAA-mediated inhibition of dopaminergic neurons
in vivo (Paladini et al. 1999
), effects that are
attributable to stimulation and blockade of presynaptic inhibitory
GABAB autoreceptors.
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CONCLUSIONS |
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Electrical stimulation of the STN gives rise to a mixed response
consisting of an EPSP and an IPSP occurring nearly simultaneously in
pars compacta dopaminergic neurons in vitro. The EPSP is mediated monosynaptically by both NMDA and non-NMDA glutamate receptors and the
IPSP is mediated mono- and disynaptically by
GABAA receptors. The IPSP arises not only
monosynaptically from co-activation of descending GABAergic fibers from
striatum and/or globus pallidus, but also disynaptically from
glutamatergic synaptic excitation of GABAergic neurons within
substantia nigra. It is likely that this is the cause of the initial
inhibition frequently reported following stimulation of the STN in vivo
(Robledo and Feger 1990; Smith and Grace
1992
). Whether activation of the STN by endogenous stimuli in
vivo also leads to mixed effects on nigral dopaminergic neurons depends
on whether single subthalamic efferent fibers synapse with both
dopaminergic and nondopaminergic neurons in substantia nigra, or
conversely, on whether different population of subthalamic efferents
which synapse selectively on dopaminergic or nondopaminergic neurons
are activated simultaneously.
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ACKNOWLEDGMENTS |
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We thank F. Shah for excellent technical assistance.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-34865.
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FOOTNOTES |
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Address for reprint requests: J. M. Tepper, Rutgers University, Center for Molecular and Behavioral Neuroscience, 197 University Ave., Newark, NJ 07102.
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 18 February 1999; accepted in final form 6 April 1999.
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REFERENCES |
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