Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark
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ABSTRACT |
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Nedergaard, S..
Regulation of Action Potential Size and Excitability in
Substantia Nigra Compacta Neurons: Sensitivity to 4-Aminopyridine.
J. Neurophysiol. 82: 2903-2913, 1999.
Slow, pacemaker-like firing is due to intrinsic membrane
properties in substantia nigra compacta (SNc) neurons in vitro. How these properties interact with afferent synaptic inputs is not fully
understood. In this study, intracellular recordings from SNc neurons in
brain slices showed that spontaneous action potentials (APs) were
attenuated when generated from lower than normal threshold. Such APs
were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and could
be related to non-N-methyl-D-aspartate
(NMDA) receptor-mediated spontaneous excitatory postsynaptic
potentials (EPSPs). The AP attenuation was reproduced by
stimulus-evoked EPSPs and by current injections to the soma. APs evoked
from holding potentials between 40 and
60 mV were reduced in width
by Cd2+ (0.2 mM). Tetraethylammonium chloride (TEA, 10 mM)
or 4-aminopyridine (4-AP, 5 mM) increased the AP width. However, at
more negative holding potentials, Cd2+ and TEA were
inefficacious, whereas 4-AP enlarged the AP, partly via induction of a
Cd2+-sensitive component. A monophasic
afterhyperpolarization (AHP), following attenuated APs, was little
affected by either Cd2+ or TEA, but inhibited by 4-AP,
which induced an additional, slow component, sensitive to
Cd2+ or apamin (100 nM). The AP delay showed a
discontinuous relation to the amplitude or slope of the injected
current (delay shift), which was sensitive to low doses of 4-AP (0.05 mM). The initial time window before the delay shift was longer than the
rise time of EPSPs. It is suggested that a 4-AP-sensitive current
prevents or postpones discharge during slow depolarization's, but
allows direct excitation by fast EPSPs. Fast excitation leads to AP
attenuation, primarily due to strong activation of 4-AP-sensitive
current. This seems to cause inhibition of the Ca2+ current
during the AP and reduction of Ca2+-dependent
K+ currents. Together, these properties are likely to
influence the excitability and the local, somatodendritic effects of
the AP, in a manner that discriminates between firing induced by the intrinsic pacemaker mechanism and fast synaptic potentials.
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INTRODUCTION |
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Neuronal responses to afferent inputs depend on
nonlinear properties provided by voltage-dependent membrane
conductances (Llinás 1988). In cells where the
conductances interact to generate spontaneous spikes, the diversity of
possible outcomes of a given stimulus could be particularly high. The
mesencepalic dopaminergic (DA) neuron is an example of such a cell
type. When recorded in vivo these neurons show a slow irregular firing
or a burst firing pattern (Clark and Chiodo 1988
;
Grace and Bunney 1984a
,b
; Nissbrandt et al.
1994
; Tepper et al. 1995
). These firing patterns
are believed to depend on afferent synaptic inputs, and evidence is
accumulating that excitatory pathways, using glutamate as
neurotransmitter (Smith et al. 1996
), are involved in
the control of firing in DA substantia nigra pars compacta (SNc) cells
(Charlety et al. 1991
; Chergui et al.
1994
; Christoffersen and Meltzer 1995
;
Overton and Clark 1992
). In the in vitro slice
preparation, however, the spontaneous activity is characterized by a
regular, pacemaker-like discharge, which has been attributed entirely
to intrinsic membrane properties (Grace and Onn 1989
;
Harris et al. 1989
; Nedergaard et al.
1993
; Yung et al. 1991
). This discrepancy is
explained if the synaptic drive on the in vitro cells is low (due to
the truncation of afferent fibers during slice preparation), and
implies that the in vivo activity could represent the integration of
both an intrinsic and an extrinsic influence on the discharge.
Electrophysiological studies from DA neurons in vitro have shown that
synaptic stimulation (Johnson and North 1992
;
Mereu et al. 1991
) or exogenous application of glutamate
agonists (Seutin et al. 1990
; Wang and French
1993
) elicits responses composed of both
N-methyl-D-aspartate (NMDA)- and non-NMDA
receptor-mediated events. However, it is not yet clear how, or to
which extent, discrete excitatory inputs interfere with the background
spontaneous activity and vice versa.
Unpublished observations in this laboratory have indicated that
pacemaker discharge involves a large variability in the size of
individual action potentials (APs) in the cell soma. The reason for
this variability is not obvious from the assumption that spike generation is at a fixed location and entirely related to an underlying slow oscillatory potential (Nedergaard et al. 1993). The
present study was undertaken to describe more closely the spontaneous AP variability and to investigate the possible contribution from afferent synaptic inputs and intrinsic properties to the regulation of
the AP.
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METHODS |
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Brain slice preparation and intracellular recording
Mesencephalic brain slices (coronal, 400 µm thick) were
prepared from albino guinea pigs of either sex (250-350 g) as
described previously (Nedergaard et al. 1993). The
animals were deeply anesthetized with chloroform in an airtight
container and killed by decapitation. The brain was removed, and a
block of tissue containing the midbrain was isolated and used for slice
preparation. Slices were transferred to a HEPES solution (see
Drugs and solutions), bubbled with 95% O2-5% CO2, and stored at room temperature for
at least 1 h before use. In the recording chamber the slice
surface was at the interface between a humidified atmosphere of 95%
O2-5% CO2 at 32-33°C and a standard
perfusion medium (see below). Flow rate was 1.5 ml/min.
Intracellular recordings were made via glass microelectrodes, filled
with 3 M potassium acetate (resistance 50-90 M), and connected to
an AXOCLAMP 2A bridge amplifier (Axon Instruments, Foster City,
CA). Signals were digitized and stored on videotape. At the end of each
experiment the electrode was retracted a few micrometers from the cell
and the extracellular potential recorded. Focal stimulation was
performed by means of a bipolar, insulated platinum wire electrode,
placed on the slice surface ventral to the recording site. Current
(100-500 µs, variable strength) was delivered by a stimulus
isolation unit (ISOLATOR 10, Axon Instruments).
Drugs and solutions
The HEPES storage solution contained (in mM) 120 NaCl, 2.0 KCl, 1.25 KH2PO4, 2.0 MgSO4, 2.0 CaCl2, 20 NaHCO3, 6.7 HEPES acid, 2.6 HEPES salt, and 10 glucose. The standard perfusion medium contained (in mM) 132 NaCl, 1.8 KCl, 1.25 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 20 NaHCO3, and 10 glucose. The following drugs were kept in stock solutions and dissolved in the perfusion medium to the final concentration immediately before use: bicuculline [10 µM, prepared in 10 mM stock (0.06 N HCl) from the free base], 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), D,L-2-amino-5-phosphonovaleric acid (APV, 50 µM), 4-aminopyridine (4-AP, 0.05-10 mM), CdCl2 (200 µM), tetraethylammonium chloride (TEA, 10 mM), and apamin (100 nM).
Data analyses
Signals were analyzed off-line on a PC computer, using SIGAVG software (CED, Cambridge, UK). AP thresholds were determined as the membrane potential at the point where the rate of depolarization started to increase above the baseline rate. The AP threshold and height were expressed in absolute voltage (extracellularly recorded voltage set to 0 mV). The threshold variation of spontaneous APs was calculated by subtracting the mean threshold from the most negative threshold, recorded in a period of 60-120 s. The AP duration was measured as the width at half-amplitude between threshold and peak. The rates of rise and fall were calculated as the average slope between 30 and 70% amplitude from threshold to peak. Spike afterhyperpolarization (AHP) measurement was made on visually identified subcomponents and the peak amplitude expressed in absolute voltage. The membrane input resistance (Rin) was calculated from the voltage deflection at the end of a 1-s negative current pulse of 0.1 nA. Unless stated otherwise, averaged data were expressed as means ± SE and Student's t-test used for statistical evaluation.
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RESULTS |
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Cell identification
Intracellular recordings were made from SNc neurons, located
lateral to the accessory optic tract. A total of 112 neurons were
selected for investigation, based on an AP width of >1.5 ms at
threshold, a slow, regular firing pattern, and a prominent sag during
hyperpolarizing current pulses, which are distinguishing criteria for
DA neurons in this preparation (Grace and Onn 1989; Yung et al. 1991
). Neurons with AP amplitudes of <45 mV
beyond threshold, or inability to fire repetitively in response to a 1-s long depolarizing current pulse, were not included. Most neurons (n = 95) fired in a slow (1.6 ± 0.9 Hz; mean ± SD), pacemaker-like rhythm during passive recording. The remaining
17 neurons were silent at rest.
Characteristics of spontaneous APs
Spontaneous AP properties were analyzed in 49 neurons. The majority of APs had similar threshold and shape and were followed by a biphasic AHP, with a fast (fAHP) and a slow (sAHP) component. In most cells, however, some of the APs did not conform to the normal characteristics. These (referred to as variable APs) were generated at low and variable thresholds (ranging from a few millivolts to >15 mV negative to the average threshold), reached less positive overshoot potentials, and had shorter duration than the average AP (Fig. 1, A and B). The AHP following the variable APs had an early peak and fast decay with no distinct sAHP (Fig. 1A). Individual APs showed a gradual decrease in overshoot and half-width in proportion to their threshold (Fig. 1C). Statistical comparison (see Table 1) between normal and variable APs [with a large (>8 mV) difference in threshold] showed a significant difference in both overshoot and half-width. The reduced width of the variable APs was reflected in a faster rate of fall and a reduced ratio of rate of rise to rate of fall. Their mean rate of rise was slightly higher than the control APs, but this difference was not statistically significant. The fAHP following the variable APs reached a similar peak potential as control APs.
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Addition of the GABAA receptor blocker bicuculline (10 µM) had no detectable effect on the discharge properties. The glutamate receptor antagonist CNQX (10 µM) applied alone, or together with APV (50 µM), caused a marked reduction of the AP variability (Fig. 2A). The threshold variation of spontaneous APs (see METHODS) in 46 cells in the absence of CNQX ranged between 1.0 and 16.7 mV (mean, 7.7 ± 0.5 mV). This value was reduced from 8.9 ± 1.3 mV to 2.7 ± 0.5 mV in seven cells exposed to CNQX (Fig. 2B). Application of APV alone (n = 6) had little and inconsistent effects on the variation in AP threshold and size (Fig. 2B).
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Spontaneous excitatory postsynaptic potentials (EPSPs)
These observations indicate that the AP shape is influenced by spontaneous EPSPs. To verify the possible existence of such potentials, voltage records sampled over several minutes were examined in each cell. Isolated transient depolarization's, with a rising slope fast enough to compare with the initial phase of full spikes, were found in 46 of the 49 cells (Fig. 3). These putative EPSPs occurred at irregular frequencies (ranging from <1 to >10 per min), had a time-to-peak between 1.0 and 3.0 ms, and varied in height in individual cells and between cells from 2 to 20 mV. They persisted during hyperpolarization below firing threshold (n = 20). The transients were not found in records of similar length in the presence of 10 µM CNQX (n = 7). A quantitative comparison between the threshold variation of APs and the transient depolarization's was not attempted, due to the large variability in frequency and size.
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Comparison of EPSP-evoked and current-pulse-evoked APs
In the presence of 10 µM bicuculline, EPSPs were produced
by focal stimulation. Discharge induced by individual EPSPs consisted of a single AP, generated at or near the EPSP peak. The EPSP rise time
(from beginning of depolarization to peak) was on average 2.8 ± 0.2 ms in 22 neurons held at membrane potentials between 60 and
70
mV (mean:
62 ± 1 mV; bicuculline 10 µM present). The
EPSP-evoked APs were attenuated and had fast decaying AHPs, both when
the membrane potential was kept stable by hyperpolarizing holding
current (Fig. 4A) and during
free firing. In the latter case, an unattenuated AP with normal AHP was
evoked in a few trials, where the stimulation coincided with a
potential close to the normal AP threshold. The degree of attenuation
(reduced overshoot and half-width) varied with the prestimulus voltage
similar to the variable APs. At
60 mV, or below, the apparent firing
threshold was typically 5-10 mV negative to normal. In the presence of
50 µM APV the EPSP rise time was unaltered, although the amplitude was sometimes slightly reduced (n = 6). The evoked APs
failed, together with a complete block of the EPSP, after application of 10 µM CNQX (Fig. 4A; n = 8).
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In the same experiments depolarizing current pulses were adjusted to
mimic EPSPs. Single APs, evoked by short (1-10 ms) pulses, resembled
EPSP-induced APs, when compared in the same cell, at similar membrane
potential (Fig. 4A). Weaker pulses, which failed to fire the
cell within ~10 ms, needed to be sustained for much longer periods
(80 ms) to evoke an AP. These long-delay APs were normal-sized and
were generated near the threshold of spontaneous APs (Fig.
4B).
Effects of ion channel blockers on the AP and AHP
Ion channel blockers were used to examine the possible
contribution of intrinsic conductances to the AP variability. To
minimize the interference from spontaneous synaptic activity, these
experiments were done in the presence of CNQX (10 µM), APV (50 µM),
and bicuculline (10 µM). Injection of constant current (positive or
negative, depending on the resting state of the cell), combined with
brief depolarizing current pulses (2-4 ms), were used to generate APs from membrane potentials between 40 and
75 mV.
The Ca2+ channel blocker
Cd2+ (200 µM) significantly reduced the
half-width of APs generated at 40 mV (n = 5). This
effect was associated with an increased rate of fall and disappearance
of the shoulder on the falling phase of the AP (Fig.
5Aa). Hyperpolarization between
40 and
60 mV led to a progressive decrease in the effect of
Cd2+. From
60 mV there was no detectable effect
on the AP duration (Fig. 5, Aa and Ba). The AP
height was not altered significantly by Cd2+ at
any potential, although a slight increase in the mean overshoot was
noted at holding potentials negative to
60 mV (Fig. 5Ba).
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In separate experiments, TEA (10 mM) caused a broadening of the AP at
40 mV (half-width increased by >100%; n = 7), and
induced a plateau-like delay during the repolarizing phase (Fig.
5Ab). The AP height was not consistently affected. The TEA
effect decreased gradually with holding potentials between
40 and
60 mV, and became undetectable negative to
60 mV (Fig. 5,
Ab and Bb, n = 7). Addition of
200 µM Cd2+ in the presence of TEA blocked the
plateau induced from
40 mV, and reduced the AP half-width to less
than control (Fig. 5Ab). Coapplication of 200 µM
Cd2+ and 10 mM TEA had no effect on the
half-width of APs generated from holding potentials negative to
60 mV
(Fig. 5Ab; n = 5).
The response to 5 mM 4-AP was tested in 11 neurons. Here, a lowering of
the spontaneous firing threshold (3-5 mV) was noted in four of seven
active neurons after exposure to 4-AP. Compared to control APs, evoked
from equal potentials, the half-width increased in all neurons exposed
to 4-AP. This effect involved a decreased rate of fall, starting from
the beginning of the repolarization, near the AP peak, and without a
distinct plateau phase as observed with TEA (Fig. 5Ac). The
effect of 4-AP persisted during hyperpolarization below 60 mV (Fig.
5, Ac and Bc). The relative increase in
half-width was between 70 and 90% at all membrane potentials
(n = 5). The AP height was largely unaltered by 4-AP at
the more depolarized potentials. However, from
60 mV the height
increased significantly in response to 4-AP, an effect that became
larger with further hyperpolarization. Thus the strong voltage
dependency of the AP overshoot, seen under control conditions, was
clearly diminished in the presence of 4-AP (Fig. 5Bc). In
three experiments where 4-AP was already present, addition of
Cd2+ caused a reduction of the AP width. The
latter effect of Cd2+ persisted at hyperpolarized
holding potentials (Fig. 5Ac).
Figure 6 shows the effects of ion channel
blockers on the AHP of individual spikes. The biphasic AHP generated
around 40 mV was highly sensitive to 200 µM
Cd2+, which reduced the amplitude of the early
peak (fAHP) and blocked the sAHP component (Fig. 6Aa). In 10 mM TEA alone, the fAHP decreased in amplitude, whereas the sAHP
persisted (Fig. 6Ab). On the other hand, 4-AP (5 mM) had
little and variable effects on this AHP; of 10 cells examined, the fAHP
did not change to any detectable degree (n = 4, example
in Fig. 6Ac), or showed a small increase or decrease (by
2-4 mV, n = 6). The variation in the differences was
not statistically significant when tested by the Wilcoxon test for pair
differences (2
> 0.05). The sAHP was not consistently altered
by 4-AP. When evoked from
60 mV, or below, the monophasic AHP, evoked
in normal medium, showed little sensitivity to
Cd2+ alone (Fig. 6Aa;
n = 5). Similarly, TEA did not alter the overall shape
of this AHP (Fig. 6Ab) and had inconsistent and
nonsignificant effects on the amplitude (unaltered in 4 of 7 cells;
decreased by 2-3 mV in 3 cells; Wilcoxon test: 2
> 0.05).
Coapplication of TEA and Cd2+ had no further
effect (Fig. 6Ab; n = 5). In contrast, 4-AP
(5 mM) induced two distinct changes in this AHP. First, the peak amplitude was markedly reduced in all cells tested (range: 5-18 mV;
n = 11). The effect was not complete, as an early AHP
was still discernible in most cells held between
60 and
70 mV (Fig. 6Ac). Second, an additional, late component developed in the
presence of 4-AP, which was reflected in a delayed decay phase and
prolongation of the AHP (10 of 11 cells). The entire AHP complex, in
the presence of 4-AP, was inhibited by subsequent application of 200 µM Cd2+ (Fig. 6Ac; n = 4). The 4-AP-induced, late AHP component was blocked by 100 nM
apamin (Fig. 6B; n = 4), and the early peak
was markedly reduced in 3 of 3 cells by 10 mM TEA, added in the
presence of 4-AP (Fig. 6B).
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Application of TEA alone caused a consistent increase in the Rin (on average by 13%, n = 7). Neither 4-AP (n = 11) nor Cd2+ (n = 5) had detectable effects on the Rin.
4-AP effect on action potential delay and threshold
As indicated above (Fig. 4), the AP attenuation seems to
rely on the depolarizing current being strong enough to give a short delay. The possibility that the delay itself is influenced by 4-AP-sensitive processes was next examined. AP delays, monitored from
the onset of positive current steps from a holding potential between
60 and
70 mV, were inversely related to the current intensity.
However, with high intensity, the delay shifted abruptly from an
average of 82 ± 8 ms to 7 ± 1 ms (n = 18;
mean holding Vm =
65 ± 1 mV;
Fig. 7A). Logarithmic plots of
delay versus current intensity showed an almost linear relationship on
either side of the delay shift; the slope on the left being
consistently smaller than on the right side of the shift (Fig.
7C, top graph). In the presence of 4-AP (0.05-10
mM, n = 15), AP delays were generally reduced (Fig.
7A) and accompanied by a lowered firing threshold (Fig.
7B). These effects were dose dependent and were already marked at the lowest 4-AP concentration used (0.05 mM; Fig.
7B). Furthermore, with doses between 0.05 and 0.5 mM, the
length of the delay shift decreased dramatically, and the delay-current relationship approximated a uniform slope (Fig. 7C). A
quantitative estimate of the delay shift at different 4-AP
concentrations was obtained in six cells (calculations made from linear
regression lines; see Fig. 7 legend). On average, the control value was
reduced by 60% in the presence of 0.05 mM, and by 93% in 0.5 mM 4-AP
(Fig. 7D, holding Vm:
65 ± 1 mV, n = 6). A further increase in dose gave a small additional effect (99% reduction obtained in 10 mM 4-AP).
For comparison, the dose dependency of the 4-AP effect on the AP
half-width was examined in the same cells at similar holding
potentials. Here, the broadening of the AP became detectable at 0.2 mM
4-AP and increased progressively with the concentration (Fig.
7D). The response showed no sign of saturation at high doses (415% increase in half-width in 10 mM 4-AP, n = 6).
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The possible dependency of the AP delay and threshold on the rate of
depolarization was tested by varying the slope of the injected current.
Under these conditions the AP delay shifted between 45 ± 1 ms and
12 ± 1 ms at an average current slope of 102 ± 15 nA/s.
(range: 58-159 nA/s; holding Vm:
65 ± 2 mV; n = 7). This delay shift was
inhibited or blocked by 4-AP, depending on the dose (0.05-10 mM;
n = 3; Fig. 8). In
control medium, the threshold of the first AP decreased gradually with
increasing current slope in the range below the critical value for
inducing the delay shift. Higher slopes gave less variation in
threshold (Fig. 8B). In the presence of 4-AP, the threshold
obtained during slow currents were markedly lowered. However, with
increased rate of depolarization the threshold approximated the control
values (Fig. 8B). Similar effects were seen in three cells
tested with ramp depolarization. The threshold of spontaneous APs,
generated at rest, was much less affected by 4-AP than the first AP
during the ramp (see Fig. 8B). In neurons activated with
square depolarizations, the average threshold of APs evoked at 5 ms
delay in control medium (
45.7 ± 1.5 mV) was not different from
those found in the presence of 5 mM 4-AP (
47.0 ± 0.7 mV,
n = 6, P > 0.05). However, at 100 ms
delay, the threshold was lowered by ~13 mV in the presence of 5 mM
4-AP (from
40.3 ± 1.3 to
53.5 ± 1.3 mV,
P < 0.01).
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DISCUSSION |
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EPSP-induced APs
In this study, SNc neurons with membrane properties characteristic
of DA cells were examined. Spontaneous APs displayed large variabilities, which decreased in the presence of CNQX. Putative non-NMDA receptor-mediated EPSPs were found in the same cells. Both
observations are in line with previous demonstration of a spontaneous
excitatory input in this preparation (Mereu et al. 1991). It is conceivable that the observed effects were due to activity of glutamatergic interneurons located within the confines of
the slice; however, evidence for the existence of such neurons is
lacking. Midbrain DA cells receive afferent glutamatergic inputs from
several external sources, including the cerebral cortex (Sesack et al. 1989
), the subthalamic nucleus (Hammond et al.
1978
; Kita and Kitai 1987
), and the
pedunculopontine nucleus (Charara et al. 1996
;
Scarnati et al. 1986
). The effects seen here could
reflect some form of spontaneous release of glutamate from terminals of cut fibers from any of these structures. Assuming that a spontaneous EPSP results from transmitter release from a single terminal or terminals of a single fiber, and that the variable APs were generated directly by such EPSPs, it seems possible that activity in one afferent
axon can be sufficient to activate the postsynaptic cell. In the light
of the constraints on spontaneous firing exerted by intrinsic membrane
conductances (Grace and Onn 1989
; Nedergaard et
al. 1993
; Shepard and Bunney 1991
), such high
excitability state seems surprising. One explanation could be that
these terminals were localized close to the AP initiation site at a
distance from the soma. Indeed, the efficacy of an input has been shown
to depend on its location relative to the initial segment of the axon,
which often emerges from a dendrite (Häusser et al.
1995
). Furthermore, dendritic APs are facilitated by EPSPs in
this preparation (Nedergaard and Hounsgaard 1996
).
Whether such effects play a significant role here is unsettled.
Stimulus-evoked EPSPs had similar, voltage-dependent effects on the AP
shape and were found to fire the cell from a lower apparent threshold
than spontaneous APs. Both effects were also seen with current pulses
in the soma. This indicates that the place of origin of the
depolarization is not the only critical factor. Alternatively, as
discussed below, the excitability could be variable, or conditional,
depending on the dynamics of certain membrane conductances.
The finding that CNQX alone blocked discharge related to both spontaneous and stimulus-evoked EPSPs indicates that non-NMDA receptors are involved in either type of stimulation.
Voltage-dependent contribution of ionic currents to the AP
Current-pulse-evoked attenuated APs has been noted previously
(Grace 1990; Grace and Onn 1989
;
Nedergaard and Greenfield 1992
), but not systematically
characterized. The contribution of different ionic currents to the AP
shape was found here to be highly dependent on the membrane potential.
Near the normal threshold of
40 mV, 200 µM
Cd2+ caused an accelerated repolarization and a
reduced AHP, which suggests that voltage-dependent
Ca2+ current underlies a depolarizing component
in the late phase of the AP and mediates activation of outward currents
involved in the AHP. The prolonged AP and reduced fAHP in the presence of 10 mM TEA indicates that the Ca2+-dependent
K+ current Ic,
and possibly a delayed rectifier (Silva et al. 1990
) contribute significantly to the AP repolarization and to the fAHP.
At 40 mV threshold the response to 5 mM 4-AP involved a slowed
repolarization with insignificant change in the AHP. A 4-AP-sensitive, A-type K+ current has previously been
demonstrated in acutely dissociated (Silva et al. 1990
)
and cultured (Liu et al. 1994
) DA neurons from the rat,
and, recently, in slices from the mouse (Bruns et al.
1998
). This current activates at potentials positive to
55 mV, and steady-state inactivation begins at
80 and is complete at
40 mV (half-maximal at
65 mV). The broadening effect found here at
40 mV threshold could therefore indicate the presence of a
4-AP-sensitive outward current, distinct from the A-type current.
Depolarization-activated K+ channels are composed
of
-subunits encoded by four gene families, Kv1-Kv4. Homomeric
channels formed by Kv1.4, 3.4, 4.1, 4.2, or 4.3 subunits display rapid
inactivation and are sensitive to 4-AP, properties similar to native
A-type currents (Baldwin et al. 1991
; Schröter et al. 1991
; Serôdio et al.
1994
, 1996
; Stühmer et al.
1989
). The Kv2.1, 2.2, 3.1, and 3.2 channels are slowly
inactivating (delayed rectifier type), as are most members of the Kv1
group [Kv1.1, 1.2, and 1.5 acquire rapid inactivation when
co-expressed with
1 subunits (Heinemann et al. 1996
;
Rettig et al. 1994
)]. The Kv3 channels are highly
sensitive to both TEA and 4-AP (Rettig et al. 1992
),
whereas Kv2 channels show intermediate sensitivity to TEA, and Kv2.1 is
also sensitive to 4-AP (at least in the rat) (Pak et al.
1991
). The involvement of Kv3 channels here is questionable, because an in situ hybridization study failed to demonstrate mRNA encoding any of the known Kv3 related proteins in rat SNc
(Weiser et al. 1994
). The lacking effects of 4-AP on the
AHP could reflect a fast deactivation rate of the 4-AP-sensitive
current. This would not compare to the relatively long deactivation
time constant characteristics of Kv1 and Kv2 channels (
15 ms)
(Grissmer et al. 1994
; Martina et al.
1998
). However, in the present study additional recruitment of
Ca2+-activated K+ current,
secondary to the AP broadening, could have obscured an effect on the
AHP. Hence it is conceivable that a 4-AP-sensitive delayed rectifier
could contribute significantly to the AP repolarization at
40 mV
threshold. Nevertheless, this issue needs further clarification because
the sustained outward current evoked from
40 mV was unaffected by 5 mM 4-AP, as reported by Silva et al. (1990)
. Their study was made on acutely dissociated cells from 5- to 7-day-old rats, which
might deviate in channel composition from the adult guinea pig.
Finally, some contribution from A-type channels at
40 mV threshold
should not be discounted, because these spikes were triggered during
ongoing spontaneous depolarizations from more negative potentials. The
time constant of inactivation of the A-type current could be
sufficiently long (between 30 and 53 ms) (Bruns et al.
1998
; Silva et al. 1990
) to leave an appreciable amount of current available for activation at the time of the AP.
A lack of effect of 5 mM 4-AP on the AP width has been reported in rat
DA neurons (Grace 1990), which is in contrast to the present findings. However, apart from a possible species difference between rat and guinea pig, these data might be incomparable, because
the APs were measured here with respect to the offset membrane
potential (given the high voltage sensitivity of the AP width), shown
to be lowered by 4-AP during free firing (Grace 1990
;
and present study).
The inefficacy of Cd2+ and TEA, seen during
hyperpolarization, indicates that Ca2+-dependent
currents and TEA-sensitive delayed rectifiers play little role in the
shaping of the AP and AHP evoked from potentials negative to 60 mV.
The accelerated repolarization and monophasic AHP emerging from
subthreshold potentials (negative to
40 mV) seems to reflect the
progressive recruitment of outward current, sensitive to millimolar
concentrations 4-AP and insensitive to 10 mM TEA. These characteristics
correlate well to the known properties of the A-type current. It is
likely that A-type channels underlie an increasing portion of the
4-AP-sensitive current at hyperpolarized potentials, and hence play a
major role in the AP attenuation. Evidence from other studies indicate
that Kv4 channels may contribute to this current: first, the rate of
inactivation is shown to be voltage independent (Silva et al.
1990
), a distinguishing characteristic of Kv4 channels
(Serôdio et al. 1994
), and, second, mRNA
transcripts encoding Kv4.3 subunits has recently been identified in the
SNc region (Serôdio and Rudy 1998
). The Kv1.4
protein is abundant in the substantia nigra, but mRNA for Kv1.4 was not
localized in this region and could originate from projecting neurons
within the striatum (Sheng et al. 1992
).
Regulation of Ca2+-dependent currents during the AP
At hyperpolarized holding potentials the broadening effect of 4-AP
involved a recruitment of Ca2+ current, because a
partial reversal of the effect was obtained after
Cd2+ application (Fig. 5). Furthermore, the AHP
evoked in the presence of 4-AP had both an early component, sensitive
to Cd2+ or TEA, and a slow component sensitive to
Cd2+ or apamin (Fig. 6). The latter finding
corresponds to the pharmacological profile of the biphasic AHP at
normal threshold (Nedergaard et al. 1993). Hence the
lack of Cd2+ sensitivity of the AP and AHP could
not be attributed to a direct effect of the hyperpolarization on the
Ca2+ current. Instead, the most likely
explanation for these findings is that the additional 4-AP-sensitive
current, activated from hyperpolarized potentials, is strong enough to
inhibit the Ca2+ current during the AP, and
thereby prevent activation of the Ca2+-dependent
K+ currents underlying the AHP. A simple shunting
effect, imposed by the 4-AP-sensitive current could also mask a
Ca2+-dependent component of the early AHP.
However, it is difficult to see how this effect alone could account for
the abolishment of the sAHP.
4-AP-sensitive delay shift
The delay shift constitutes a temporal separation between normal
and attenuated APs, because the latter were confined to the initial
time window (Fig. 4). The sensitivity to 4-AP of AP delays as long as
1 s indicates that a slowly inactivating current counteracts depolarization in a voltage range below firing threshold. This current
is responsible for the delay shift. The higher sensitivity to 4-AP of
the delay shift compared with the AP width (Fig. 7D) could
indicate that these two parameters show a large difference in their
dependency of the amount of 4-AP-sensitive current available. Alternatively, separate currents may coexist, with different
sensitivity to 4-AP. Transient currents, distinguished from the
IA by a slow inactivation and high
sensitivity to 4-AP, has been shown in different neurons to mediate
delayed excitation (McCormick 1991; Nisenbaum et
al. 1994
; Spain et al. 1991
; Storm
1988
).
Role of 4-AP-sensitive current in regulation of AP threshold
The threshold of the first AP was lowered by 4-AP depending
on the rate of depolarization. This suggests that the effect of the
4-AP-sensitive current on the AP delay can, at least partly, be
attributed to an increased threshold of APs generated at long delays.
Conversely, the threshold of short-latency APs, generated within the
initial time window before the delay shift, was unaffected by 4-AP. The
latter observation can be explained if the activation kinetics of the
4-AP-sensitive current is slow at subthreshold potentials, and
therefore has little influence on the initiation of an early AP.
Considering that the early APs were precisely the ones that were
maximally attenuated by the 4-AP-sensitive current, it would seem that
the rate of activation increases markedly during the spike. In fact,
the activation time constant of the A-current in these neurons is
reported to increase from 4.5 to 0.9 ms in the voltage range between
55 and
15 mV (Bruns et al. 1998
). This finding
seems, at least qualitatively, to be in accordance with the above interpretation.
With 4-AP present, the AP threshold decreased with increasing delay
(decreasing rate of depolarization). The reason for this was not
pursued here. However, such a relationship would be expected in a cell
where the site of spike initiation is at an electrotonic distance from
the recording site, due to the lower degree of electrotonic filtering
of slow potentials. As reported by Grace (1990), the threshold of the initial segment (IS) component of the AP, as seen from
the soma, is particularly sensitive to 4-AP, possibly due to the
location of a 4-AP-sensitive conductance in the segment of dendrite
between the IS and the soma. The present results seem compatible with
this interpretation and indicate further that activation of this
conductance may obscure and oppose the frequency-dependent variation of
passive filtering.
Functional implications
The delay shift is likely to be exploited under
physiological conditions, because the EPSP rise time (2.8 ms) was well
below the lower limit of the shift (7-12 ms). The high efficacy of
EPSPs at hyperpolarized potentials could be due to their ability to depolarize the membrane rapidly and thereby "escape" the delay shift. Judged from the effect of ramp injections, a somewhat slower depolarization would fail to cause direct excitation, unless sustained for several tens of milliseconds. It seems that this unlinear response
property would function as a filter discriminating between fast and
slow events. An obvious consequence of such a mechanism is that fast
EPSPs are allowed to mediate precisely timed outputs (in millisecond
scale) with respect to presynaptic discharge. Because direct AP
generation could occur over the range of membrane potentials (40 to
65 mV) normally experienced by the cell during spontaneous firing,
such activation might be more or less independent on the background
activity. The pacemaker potential is rising slowly compared with an
EPSP, which may explain why APs in the presence of CNQX are unlikely to
be fired from hyperpolarized thresholds (Fig. 2). Therefore a
discriminatory function based on the slope could be an effective way of
keeping a high sensitivity to fast afferent signals, while at the same
time maintaining a low intrinsic firing rate. The influence of afferent
connections on the discharge may involve both a direct spike activation
by fast EPSPs and an indirect modification of the ongoing activity by
slow inputs. The shifts in firing patterns of nigral neurons, seen to
be implicated in changes in motor behavior (Romo and
Schultz 1990
), could be a reflection of transitions
between slow and fast afferent drive.
In SNc cells the AHP influences the firing properties, in
particular the sAHP component, which has a dampening and regularizing effect on the discharge (Shepard and Bunney 1991).
Inhibition of the sAHP seems, from this study, to be a general
consequence of activating these cells from low membrane potentials. The
exact role of this inhibition for the firing properties under various conditions was not examined. It is possible that a shortened AHP could
enhance the excitability during repetitive activation. This would
further dissociate the properties of EPSP-driven discharge from the
intrinsic pacemaker mechanism.
Calcium influx during the AP may have several functions. Because the
Ca2+ component of the AP causes prolongation of the
depolarizing phase, it may help backpropagation of unattenuated APs
into the dendrites as suggested by Häusser et al.
(1995). Consequently, inhibition of the Ca2+
component could reduce the dendritic AP and perhaps shorten the distance over which it propagates. Attenuated APs, driven by EPSPs, may
therefore be less effective in invading the dendrites than intrinsic
APs. Dendritic voltage-dependent Ca2+ conductances
(Hounsgaard et al. 1992
), co-activated during the AP,
may influence intracellular processes, including those involved in
dendritic release of dopamine (Geffen et al. 1976
;
Rice et al. 1994
, 1997
). Several studies
have shown a lack of correlation between the discharge rate and
dopamine release in substantia nigra (Cheramy et al.
1981
; Nieoullon et al. 1977
; Nissbrandt et al. 1985
), which questions the role of APs in the release
process. However, a discrepancy between the discharge rate and the
amount of released dopamine might be expected, if dendritic
Ca2+ influx depends on the AP size. It is possible that
dendritic dopamine release, for this reason, is facilitated or
inhibited, depending on the mode of spike activation, rather than on
the overall discharge rate.
In conclusion, this study suggests that the excitability and action
potential size are regulated during excitatory synaptic transmission,
depending on activation of 4-AP-sensitive current in the postsynaptic
cell. This current may be composed of both delayed rectifier-type and
A-type current, the latter of which becomes dominant at subthreshold
membrane potentials. A regulatory role of IA
on the membrane response properties has previously been shown in
cerebellar Purkinje cells and hippocampal pyramidal neurons
(Andreasen and Lambert 1995; Hoffman et al.
1997
; Midtgaard 1994
), where its dampening
effect on dendritic excitability appears to be a means by which
synaptic integration can be regulated locally. The present study adds
to these findings and suggests that another important function of such
current is to provide spontaneously active neurons with the ability to
discriminate between different afferent inputs and their own intrinsic activity.
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ACKNOWLEDGMENTS |
---|
I thank Drs. M. Andreasen and J. Hounsgaard for helpful comments on the manuscript.
This work was supported by a grant from the Aarhus University Research Foundation.
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FOOTNOTES |
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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 7 December 1998; accepted in final form 23 July 1999.
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REFERENCES |
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