Department of Physiology, Faculty of Medicine and Health Science,
University of Auckland, Auckland, New Zealand
 |
INTRODUCTION |
Dopaminergic
neurons of the Substantia Nigra zona compacta (SNc) project to striatum
where they control neurons involved in the execution of motor programs
(e.g., Pucak and Grace 1994
). The SNc neurons receive
glutamatergic inputs from the prefrontal cortex, the subthalamic
nucleus, and the pedunculopontine tegmental nucleus (Kitai et
al. 1999
), which normally regulate the firing of these neurons
through both N-methyl-D-aspartate (NMDA) and non-NMDA receptors (e.g., Chergui et al. 1993
;
Christoffersen and Meltzer 1995
). There is also evidence
that increased glutamatergic input may contribute, through
exitotoxicity, to degeneration of SNc neurons in Parkinson's disease
in humans or related animal models (Blandini et al.
1996
) and that the hyperactive input to neurons that survive
neurodegeneration may lead to some symptoms of the disease (e.g.,
Rodriguez et al. 1998
).
Recent studies indicate that CNS glutamatergic synapses not only
control neural activity and mediate excitotoxic injury but also
determine neuron survival during the pre- and postnatal periods through
activation of the NMDA receptors (see DISCUSSION for
references). However, the expression of these receptors in the
postsynaptic membrane of SNc neurons in the early stages of development
remains uncertain. In a recent electrophysiological study, Wu
and Partridge (1998)
were unable to demonstrate any
NMDA-induced currents in neurons isolated from 2-wk-old rats, a
postnatal period associated with a transient peak in naturally
occurring SNc cell death (Janec and Burke 1993
;
Oo and Burke 1997
). Since downregulation of NMDA receptor expression may have important consequences for neuronal survival during development, we felt it was important to re-examine the
presence of functional NMDA receptors in SNc of young rats.
 |
METHODS |
Experiments were conducted on SNc neurons acutely dissociated
from P4 to P16 Wistar rats. Rat pups (<8 days old) were anesthetized by hypothermia, and 0.6 µl injections of 0.5% Fluoro-Gold
(Fluorochrome) were made bilaterally into striatum (cf. Silva et
al. 1990
). Neuron dissociation was conducted using a protocol
similar to that applied by us previously to isolate cells from the
medulla oblongata (Lipski et al. 1998
). In brief, 2-9
days after injection of the retrograde label, rats were anesthetized
with CO2 and decapitated. The brains were removed
and the midbrain region cut transversely with a Vibratome (200 µm).
The sections containing SNc were mildly digested with papain (20 U/ml
10-15 min, 32°C; Worthington), the SNc region dissected out using a
scalpel blade, and the tissue gently triturated with fire polished
Pasteur pipettes. Cells were plated on poly-L-lysine-coated coverslips that were placed in a recording chamber (volume, 0.4 ml)
mounted on an inverted microscope equipped with fluorescence attachment
(filter block: excitation, 355-425; dichroic mirror, 455; barrier, 460 nm). The chamber was perfused (~0.4 ml/min) at room temperature
(22-24°C) with a solution containing (in mM) 150 NaCl, 3 KCl, 2.4 CaCl2, 10 HEPES, 0.01 glycine, and 15 glucose (pH
7.4). Magnesium ions were omitted unless stated otherwise. Fluoro-Gold
labeled cells were typically multipolar or oval in shape (long axis,
25-40 µm) and had several (3-6) truncated dendrites. In one
experiment, dissociated cells were fixed in 4% formaldehyde and
examined for tyrosine hydroxylase (TH) immunoreactivity using a
monoclonal TH antibody (Boehringer Mannheim) and Texas Red-labeled secondary antibody (cf. Lipski et al. 1998
). Over 90%
of Fluoro-Gold labeled neurons were also TH immunoreactive (Fig. 1,
C and D).

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Fig. 1.
Dissociated Substantia Nigra zona compacta (SNc) neurons under
phase-contrast (A and B), identified as
projecting to the striatum by Fluoro-Gold (C), and
labeled with anti-tyrosine hydroxylase (TH) antibody (D; the
same pair of neurons as in C). Scale bars: 50 µm
(A and B), 20 µm (C and
D).
|
|
Whole cell recordings were made using a conventional tight-seal, or
perforated-patch, configuration. Conventional recordings (seal, >10
G
) were made with pipettes filled with a solution containing (in mM)
130 KF, 5 NaCl, 11 EGTA, 10 HEPES, 1 CaCl2, 10 glucose, and 3 Na ATP or 140 CsF, 10 tetraethylammonium chloride, 10 HEPES, 5 EGTA, 10 glucose, and 3 Na ATP (pH = 7.25). For
perforated-patch recording (access resistance, <20 M
), the pipette
solution contained (in mM) 125 potassium methanesulphonate, 20 KCl, 15 NaCl, 7.5 HEPES, and 2 EGTA, pH 7.3, with Amphotericin B (200 µg/ml).
Current- and voltage-clamp recordings were made with Axopatch 200B
amplifier and the pCLAMP software (Axon Instruments). Drugs (NMDA,
dopamine, and a competitive NMDA receptor antagonist
2-amino-5-phosphopentanoate, APV; all from Sigma) were dissolved in the
"external" solution and applied through a multibarrel pressure
ejection pipette positioned ~50 µm away from the examined cell (1- to 5-s injections; 6-8 psi; interval, 1 min).
 |
RESULTS |
The following criteria were used to select neurons for testing
with NMDA: healthy appearance (smooth plasma membrane, bright under
phase-contrast, lack of signs or swelling or shrinkage, dendrites
without "beading"; Fig. 1, A and B); the
presence of retrograde labeling after striatal injection of Fluoro-Gold
(Fig. 1C); hyperpolarization-induced time-dependent inward
(Ih) current under voltage clamp or a
depolarizing "sag" in current clamp (both are characteristic of
these neurons) (e.g., Silva et al. 1990
; Washio
et al. 1999
) (Fig. 2,
B and C); the
ability to fire repetitive action potentials in the current-clamp mode
in response to depolarizing current (Fig. 2C) (Silva
et al. 1990
; Yung et al. 1991
); and inhibition of the firing (in animals older than P9) by application of 50 µM
dopamine (Fig. 2D) (Silva et al. 1990
;
Washio et al. 1999
). Most cells showed periods of
spontaneous regular firing, with action potentials (duration, 4.9 ± 0.3 ms; n = 25) preceded by slowly rising
depolarizations (indicative of pacemaker potentials; see Fig.
2C) and were followed by long-lasting
afterhyperpolarizations (Fig. 2C) (Grace and Onn
1989
; Yung et al. 1991
).

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Fig. 2.
Electrophysiological properties of an isolated SNc neuron.
A: fast inward (Na+) and slow outward
(K+) currents in response to voltage steps.
B: Ih current evoked by a
step command from 60 to 130 mV. C and
D: potentials recorded in response to current pulses
(C) and to close-cell application of dopamine
(D; 50 µM).
|
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A total of 29 cells (14 from P4 to P9 and 15 from P10 to P16 animals)
fulfilled the preceding criteria and were examined for responses to
NMDA application (100 µM; 1 s). In the voltage-clamp mode
(Vhold =
60 mV), all cells responded
with an inward current that reached a peak amplitude near the end of
injection (Fig. 3A). When
depolarizing voltage ramps were used (together with Cs-based internal
solution), the current reversed in polarity at around 0 mV
(n = 3, Fig. 3C2). In current-clamp, NMDA
induced membrane depolarization and high-frequency firing (or
depolarizing block of firing; not illustrated) in all 20 tested cells
from both age groups (Fig. 3B). The peak amplitudes of the
responses, together with the values of the membrane capacitance
(indicative of cell size) and a measure of the
Ih current, are given in Table 1. No statistically significant
difference in the peak amplitude of the NMDA-induced inward current, or
the membrane depolarization, was found in neurons from the two examined
age groups. The inward current could be blocked by co-application of
200 µM APV (n = 5, Fig. 3A). It was also
strongly reduced or abolished in the presence of 1 mM
Mg2+. The effect was voltage dependent
(n = 3; Fig. 3C, 1 and 2). Responses to NMDA application were observed in all cells tested with
the conventional (n = 24) or perforated-patch
(n = 5) recording technique.

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Fig. 3.
Responses of 2 SNc neurons to close-cell application of
N-methyl-D-aspartate (NMDA, 100 µM).
A: inward current blocked by APV (200 µM; added both
to the "external" and "injection" solutions). B:
potentials recorded in the same neuron during current-clamp ( 12 pA
was used to stop spontaneous firing). C1:
I-V relationships obtained in another neuron using 1 s
depolarizing ramps during control and near the peak of NMDA-induced
current (5 s injections) in the presence and absence of
Mg2+. C2: voltage dependence of NMDA evoked
current (after subtraction of appropriate traces in C1).
KF- and CsF-based "internal" solutions were used for recordings
illustrated, respectively, in A, B, and C,
1 and 2 (C, 1 and
2, corrected for the liquid junction potential; 7.5 mV).
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 |
DISCUSSION |
The expression of NMDA receptors in adult rat dopaminergic SNc
neurons has been documented with receptor binding studies (Albin et al. 1992
) and molecular identification of receptor subunits (particularly the NMDAR1 and R2C) (Albers et al. 1999
).
These receptors are believed to regulate the firing rate and the
bursting pattern of activity of SNc neurons (e.g., Chergui et
al. 1993
; Christoffersen and Meltzer 1995
;
Johnson et al. 1992
), and to modulate release of
dopamine both from the somato-dendritic region and presynaptic
terminals in the striatum (Araneda and Bustos 1989
;
Cheramy et al. 1996
). To our knowledge, there are no
data on NMDA receptor expression (using ligand binding, RNA or protein analysis) in rats under the age of P14. Our electrophysiological results demonstrate the presence of functional NMDA receptors in SNc
neurons isolated during this early postnatal period. The NMDA-induced
responses were postsynaptic (i.e., evoked in the soma and/or proximal
dendrites) as, due to acute cell isolation, any presynaptic effects
were eliminated. It is uncertain why our results differ from those
published by Wu and Partridge (1998)
, who concluded that
glutamate depolarizes SNc neurons in 2-wk-old rats only by non-NMDA
receptors. It is unlikely that the difference is due to various enzymes
used for cell isolation (papain versus pronase), as Wu and
Partridge (1998)
did observe NMDA-induced currents in cells
isolated from an adjacent region. The SNc region is not entirely
homogenous with respect to axonal projections and also contains
nondopaminergic neurons (Yung et al. 1991
). Therefore
the different findings could be explained by the fact that we used more
strict criteria for identification of SN neurons as dopaminergic and
projecting to the striatum, systematically identifying the cells by
retrograde labeling, the presence of the characteristic
Ih current, and the response to dopamine.
Unless "stem cells" are present in SNc in mature animals (see
Janson et al. 2000
), the final number of neurons present
in this nucleus in mature animals is determined by the proportion of
cells that are not "trimmed out" by physiological apoptosis during
the early stages of development (Jackson-Lewis et al.
2000
). Studies conducted in rodents revealed that a major
natural cell death event takes place in SNc between E20 and P8
(Jackson-Lewis et al. 2000
; Janec and Burke
1993
; Oo and Burke 1997
). Interestingly, there
is a second peak of apoptosis at around P14. The basis for the second,
postnatal apoptotic peak in cell death is unclear. One possibility is
that the peak is correlated with competition for synaptic contacts
(Oppenheim et al. 1991
) because the greatest increase in
number of synapses in the striatum occurs between P13 and P17
(Hattori and McGeer 1973
). It can also be hypothesized that it is due to a transient downregulation of NMDA receptors in SNc
neurons and the lack of the survival-promoting effect of NMDA receptor
activation. Previous studies have demonstrated that synaptic activation
of NMDA receptors by glutamate promotes neuronal survival in widespread
brain regions during development and that blockade of these receptors
triggers apoptotic neurodegeneration (Gould et al. 1994
;
Ikonomidou et al. 1999
). Our results, in contrast to the
findings of Wu and Partridge (1998)
, show consistent
expression of functional NMDA receptors in the SNc during the second
postnatal week and therefore argue against such a mechanism. In fact,
in the first weeks of postnatal life, NMDA receptors often undergo a
period of hypersensitivity rather than downregulation
(Ikonomidou et al. 1989
).
Address for reprint requests: J. Lipski, Dept. of Physiology, Faculty
of Medical and Health Sciences, The University of Auckland, Private Bag
92019, Auckland, New Zealand (E-mail:
j.lipski{at}auckland.ac.nz).