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The Journal of Neuroscience, November 15, 2000, 20(22):8493-8503
Intrinsic Membrane Properties Underlying Spontaneous Tonic Firing
in Neostriatal Cholinergic Interneurons
Ben D.
Bennett1,
Joseph
C.
Callaway2, and
Charles J.
Wilson1
1 Cajal Neuroscience Research Center, Division of Life
Sciences, University of Texas, San Antonio, Texas 78249 and
2 Department of Anatomy and Neurobiology, University of
Tennessee, Memphis, Tennesse 38163
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ABSTRACT |
Neostriatal cholinergic interneurons produce spontaneous tonic
firing in the absence of synaptic input. Perforated patch recording and
whole-cell recording combined with calcium imaging were used in
vitro to identify the intrinsic membrane properties underlying endogenous excitability. Spontaneous firing was driven by the combined
action of a sodium current and the hyperpolarization-activated cation
current (Ih), which together ensured
that there was no zero current point in the subthreshold voltage range.
Blockade of sodium channels or Ih
established a stable subthreshold resting membrane potential. A
tetrodotoxin-sensitive region of negative slope conductance was
observed between approximately  60 mV and threshold (approximately
50 mV) and the h-current was activated at all subthreshold voltages.
Calcium imaging experiments revealed that there was minimal calcium
influx at subthreshold membrane potentials but that action potentials
produced elevations of calcium in both the soma and dendrites.
Spike-triggered calcium entry shaped the falling phase of the action
potential waveform and activated calcium-dependent potassium channels.
Blockade of big-conductance channels caused spike broadening.
Application of apamin, which blocks small-conductance channels,
abolished the slow spike afterhyperpolarization (AHP) and caused a
transition to burst firing.
In the absence of synaptic input, a range of tonic firing patterns are
observed, suggesting that the characteristic spike sequences described
for tonically active cholinergic neurons (TANs) recorded in
vivo are intrinsic in origin. The pivotal role of the AHP in
regulating spike patterning indicates that burst firing of TANs
in vivo could arise from direct or indirect modulation of the AHP without requiring phasic synaptic input.
Key words:
TANs; tonic firing; rhythmic bursting; spike sequences; irregular firing; sodium current; Ih
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INTRODUCTION |
Single-unit recordings from awake,
behaving primates have been used extensively to investigate the
response properties of neostriatal cholinergic interneurons. The
cholinergic cells are identified by their ongoing spiking activity and
are consequently referred to as tonically active neurons (TANs). In
contrast to spiny projection cells, TANs exhibit a pause in firing,
which is phase-locked to sensory stimuli that trigger learned and
rewarded movements (Crutcher and DeLong, 1984 ; Kimura et al., 1984;
Liles, 1985; Schultz and Romo, 1988; Hikosaka et al., 1989;
Apicella et al., 1991; Aosaki et al., 1994a,b, 1995; Raz et al., 1996; Watanabe and Kimura, 1998). The pause response emerges as the task is
learned, which has led to the suggestion that the brief cessation in
firing represents a physiological correlate of motor learning within
the neostriatum (Graybiel et al., 1994). After systemic MPTP
administration and the emergence of parkinsonian symptoms, the tonic
firing of TANs is replaced by persistent oscillatory activity (Raz et
al., 1996), and the pause response can no longer be triggered by
sensory stimuli (Aosaki et al., 1994a). Together, these data suggest
that spike timing in cholinergic cells is critically involved in both
the normal functioning of the neostriatum and the pathophysiological
processes that occur in parkinsonian states.
Intracellular in vivo recordings from neostriatal
cholinergic interneurons have demonstrated that summation of only a few depolarizing synaptic potentials is sufficient to trigger spiking (Wilson et al., 1990). These data suggested that synaptic inputs are
the principal determinants of action potential timing in cholinergic cells and have been interpreted as evidence that the tonic, irregular firing pattern of TANs recorded in vivo reflects the
temporal structure of the synaptic barrage (Wilson, 1993; Aosaki et
al., 1995; Yan and Surmeier, 1997; Bennett and Wilson, 1998; Watanabe and Kimura, 1998). However, spontaneous firing has been commonly observed in cholinergic cells under conditions of reduced synaptic input both in vivo (Wilson et al., 1990) and in
vitro (Bennett and Wilson, 1998; Calabresi et al., 1998; Lee et
al., 1998). More recently, we found that, after pharmacological
blockade of synaptic activity in vitro, cholinergic cells
continued to fire, exhibiting spiking rates and patterns very similar
to those of TANs recorded in vivo (Bennett and Wilson,
1999). It therefore seems likely that cholinergic cells possess ionic
currents that confer endogenous excitability and that the spiking
pattern of TANs reflects an interaction between synaptic input and the
intrinsic properties of these cells. An understanding of the intrinsic
mechanisms that are involved in spike generation and patterning is
central to determining the origin of the pause response detected during
unit recordings from TANs in vivo. Furthermore, the
possibility that oscillatory activity observed in parkinsonian states
might arise from alterations of the intrinsic membrane properties has
potential clinical relevance because of the known efficacy of
cholinergic antagonists in the treatment of Parkinson's disease
(Hingtgen and Siemers, 1998; Schrag et al., 1999).
The objectives of this study were therefore to elucidate the intrinsic
mechanisms that contribute to tonic firing and to investigate the
consequences of perturbation of the different ionic currents that are
involved in spike generation and patterning. With the exception of the
calcium imaging experiments, the perforated-patch configuration was
used throughout to avoid alteration of the electrical properties of
cholinergic cells that can occur during conventional whole-cell
recording (Bennett and Wilson, 1999).
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MATERIALS AND METHODS |
Slice preparation. Brain slices were prepared using
previously described procedures (Bennett and Wilson, 1998, 1999).
Briefly, postnatal day 21-25 Sprague Dawley rats of either sex were
deeply anesthetized with ketamine-xylazine and perfused via the
ascending aorta with 30-50 ml of ice-cold (4°C) sucrose-substituted
(Aghajanian and Rasmussen, 1989) saline containing (in
mM): 230 sucrose, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 10 MgSO4, and 10 glucose. Coronal slices of 300 µm thickness were then cut through the
neostriatum with a vibrating blade microtome (Leica, Deerfield, IL) and
transferred to a holding chamber containing artificial CSF (ACSF)
(25 ± 2°C) of composition (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 2 MgSO4, and 10 glucose. The ACSF was continuously oxygenated and recording commenced 1 hr after the slicing procedure.
Visualized recording. Slices were transferred to the
recording chamber and continuously perfused (2-3 ml/min) with
oxygenated ACSF (35 ± 1°C). Neurons within the neostriatum were
visualized using infrared differential interference contrast
videomicroscopy (Dodt and Zieglgansberger, 1990; Stuart et al., 1993).
Cholinergic cells were selected for recording on the basis of their
characteristic somatodendritic morphology, and their identity was
confirmed by their stereotyped responses to current injection (Jiang
and North, 1991; Kawaguchi, 1992, 1993; Götz et al., 1997;
Bennett and Wilson, 1998, 1999; Calabresi et al., 1998; Lee et al.,
1998; Pisani et al., 1999, 2000). Recordings were obtained using the
perforated-patch technique (Horn and Marty, 1988; Korn and Horn, 1989)
to avoid artifactual changes in the electrical properties of
cholinergic cells that can occur during whole-cell recording (Bennett
and Wilson, 1999). Gramicidin was used as the pore-forming agent (Myers and Haydon, 1972) because this was found to produce reliable
perforation and long-lasting, stable recordings (Akaike and Harata,
1994; Rhee et al., 1994; Ebihara et al., 1995; Kyrozis and Reichling, 1995). Patch electrodes were prepared from thick-walled, unfilamented borosilicate glass (Warner Instruments, Hamden, CT) on a P-87 Brown-Flaming electrode puller (Sutter Instruments, Novato, CA). The
tips of the patch pipettes were filled with a solution containing (in
mM): 119 K-MeSO4, 12 KCl, 1 MgCl2, 1 EGTA, 0.1 CaCl2,
10 HEPES, 0.4 Na-GTP, and 2 Mg-ATP, pH 7.4 (280-300 mOsm) and then
back-filled with the same solution containing gramicidin (100 µg/ml)
(Ebihara et al., 1995) yielding electrode resistances of 2-5 M .
Minimal positive pressure was applied to the pipette while it was
placed on the somatic membrane, and then slight negative pressure was used to form a seal. Only cases in which the initial seal resistance exceeded 1 G were used, and all other recordings were immediately abandoned. The electrode capacitance was compensated, but the series
resistance and whole-cell capacitance were not. The time course for
perforation varied between individual cells, but typically, data
collection commenced 15-30 min after seal formation when the series
resistance (Rs) was 53 ± 25 M
(mean ± SD; n = 61). Recordings were terminated
45 min to 3 hr after seal formation with a final
Rs of 41 ± 19 M (mean ± SD; n = 61). Sudden changes in
Rs were taken to indicate that the
patch had ruptured, and these recordings were discarded. Data were
collected using an Axopatch 200B amplifier and pClamp6 software (Axon
Instruments, Foster City, CA). Current-clamp recordings were only
obtained using the "fast CC" mode of the amplifier, which produces
voltage recordings that are comparable with those obtained with
conventional current-clamp amplifiers (Magistretti et al., 1996).
Current recordings were only undertaken when
Rs < 40 M and were restricted to
measuring steady-state currents (<100 pA) at the end of 1 sec voltage
steps (see Results). Pooled data from voltage-clamp recordings were only examined for a relatively narrow voltage region to minimize both
voltage errors attributable to the
Rs and space-clamp errors attributable
to axial current flow (Mainen et al., 1995; White et al., 1995).
Signals were filtered at 2 or 5 kHz and digitized at 10 or 20 kHz, respectively.
Calcium imaging. All procedures were as described above,
except that the whole-cell configuration was used and the patch
electrodes were filled with a solution containing (in
mM): 119 K-MeSO4, 12 KCl, 1 MgCl2, 10 HEPES, 0.4 Na-GTP, 2 Na-ATP, and
0.05-0.2 K-fura-2, pH 7.4 (280-300 mOsm). Electrical and optical data
were collected simultaneously using a Neurodata IR283 amplifier (Cygnus
Technology Inc., Delaware Water Gap, PA) and a cooled EEV37 CCD camera
(Photometrics, Tucson, AZ) running in frame transfer mode.
Current-clamp recordings were digitized at 10 kHz, and frame rates of
18-40 Hz were used for collection of optical data. Fluorescence
changes were measured using an excitation wavelength of 380 nm and were
corrected for bleaching and autofluorescence. During steady
hyperpolarization, fluorescence measurements were collected, filtered
at 3 Hz, and subtracted from episodes during which the cell was
depolarized or allowed to fire spontaneously to correct for bleaching.
Autofluorescence was corrected by subtracting the fluorescence of a
region of the slice that was devoid of fura-containing structures from
the initial value of F. Ratiometric data were not collected,
and all fluorescence measurements are therefore presented as changes in
% F/F. At an excitation wavelength of 380 nm,
fura-2 fluorescence decreases with increasing calcium concentration,
but for the sake of clarity, the data plotted are actually
%F/F so that the decreases in fluorescence
have the same polarity as increases in calcium concentration. There is
a very nearly linear relationship between
%F/F and calcium concentration for changes of
<50% F/F (Lev-Ram et al., 1992), and all
measurements in this study were within this range.
Drugs. All drugs were obtained from Sigma (St. Louis, MO)
unless otherwise noted and were applied by bath perfusion. Tetrodotoxin (TTX) (1 µM) was used to block sodium channels.
The hyperpolarization-activated cation current
(Ih) was blocked with cesium (3 mM). Because ZD7288 (100 µM; Tocris Cookson, Ballwin, MO) was found to
cause significant spike broadening, it was only used to block
Ih in experiments undertaken in the
presence of TTX. Calcium channel blockade was achieved by either
substituting cobalt (2 mM) for calcium in the ACSF or by using cadmium (400 µM). In both
cases, NaH2PO4 was omitted
from the ACSF to avoid precipitation. Cadmium and cobalt produced
indistinguishable effects, and the data were therefore pooled (see
Results). Big-conductance calcium-activated potassium (BK) channels
were blocked using either tetraethylammonium chloride (TEA) (1 mM) or iberiotoxin (100 nM), which produced identical effects, and the
data from the two groups were pooled (see Results). Small-conductance
calcium-activated potassium (SK) channels were blocked using apamin
(100 nM). Synaptic blockers were not used because
neostriatal cholinergic interneurons receive minimal synaptic input
in vitro, and these inputs have an undetectable effect on the spontaneous firing rates and patterns exhibited by these cells (Bennett and Wilson, 1999).
Data analysis. The firing rate was determined from a 2 min
sample of spontaneous activity. The coefficient of variation (CV) (SD/mean) was calculated for all cells that exhibited a mean firing rate  0.5 Hz. Spike threshold was defined as the point at which the
first derivative of voltage with respect to time exceeded 4 Vs 1 and corresponded to a
sharp inflection in the voltage trace. The mean subthreshold voltage
(Vm mean) was calculated from a 1 min
sample of the voltage trace using only points negative to action
potential threshold. Spike width was measured at threshold. The
afterhyperpolarization (AHP) amplitude was defined as the difference in
voltage between spike threshold and the peak negativity after a spike.
The AHP time-to-peak was calculated as the time difference between
threshold and the subsequent AHP peak. For cases in which drug
treatments resulted in membrane hyperpolarization, direct-current
(DC) current injection (10-60 pA) was used to reinstate the
predrug firing rate or membrane potential (in the case of quiescent
neurons). The junction potential was measured directly by recording the
voltage offset produced by sequentially immersing a patch electrode in
the electrode solution followed by ACSF. Whole-cell recordings were
corrected off-line by subtracting the measured 5 mV potential.
Comparison of corrected whole-cell data and perforated patch recordings
failed to reveal a significant difference in action potential threshold
(measured as described above), indicating that the junction potential
was negligible in the perforated patch configuration. Statistical
analyses were applied using the paired-sample t test, and
significance was assigned when p < 0.005. Data are
given as mean ± SD unless otherwise stated.
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RESULTS |
Data analysis was restricted to cholinergic cells that exhibited a
tonic single spike firing pattern or that were silent. Although
cholinergic cells that displayed spontaneous bursting, defined as
clustered spiking interrupted by large subthreshold voltage
fluctuations (Bennett and Wilson, 1999), were also encountered, they
were excluded from the present study to focus the experiments on the
ionic mechanisms underlying tonic firing. The majority (46 of 61) of
cholinergic cells exhibited spontaneous spiking (Fig.
1A), which persisted
throughout the course of a perforated patch recording, suggesting that
this technique is effectively noninvasive. Small-amplitude voltage
steps (2 mV, 50 msec) were used to assess the initial seal resistance
and to periodically determine the series resistance during a recording
(Fig. 1B). A range of firing rates (1.53 ± 1.48 Hz; range, 0.0-6.45 Hz; n = 61) and patterns (CV,
0.34 ± 0.25; range, 0.09-1.08; n = 39) were
observed during perforated patch recordings (Fig.
1C,D). A clear relationship between the firing
rate and regularity of the spike train was seen (Fig.
1D), as has been described for the spike trains of
cholinergic neurons recorded using cell-attached and extracellular
recording techniques (Bennett and Wilson, 1999), providing support for
the suggestion that perforated-patch recording was effectively
noninvasive. The mean subthreshold membrane potential was 62.3 ± 4.0 mV (n = 60), and the action potential threshold and width were 49.0 ± 3.8 mV (n = 60) and
2.5 ± 0.6 msec (n = 47), respectively.
Cholinergic cells exhibited a large slow spike AHP with a peak
amplitude of 18.0 ± 4.1 mV (n = 41) and a
time-to-peak of 53.1 ± 13.8 msec (n = 38).
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Figure 1.
Firing rates and patterns of cholinergic cells.
A, Example of a spike train recorded from a tonically
active cholinergic neuron using the perforated patch configuration 45 min after seal formation. B, Averaged responses (50-100
trials) to a 2 mV voltage step recorded immediately after seal
formation and 75 min later. C, The firing rates of
cholinergic cells were skewed toward lower values with the mean and
median falling between 1 and 2 Hz. D, A plot of
the CV versus the firing rate indicates that cholinergic cells
that fire more rapidly exhibit more regular spike trains.
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Sodium
To investigate the role of sodium influx in the generation of
tonic firing, we first examined the effect of sodium channel blockade
on spontaneous spiking. Application of TTX (1 µM) blocked action potentials (n = 21) and failed to reveal any
subthreshold oscillation in the membrane potential (Fig.
2A,B).
The observation that action potential threshold was 48.4 ± 4.2 mV before blockade of sodium channels whereas the mean membrane
potential was 59.5 ± 6.1 mV after application of TTX
(n = 21) suggests that sodium channels are activated in
the subthreshold voltage range and participate in bringing the neuron
to spike threshold. To investigate this possibility, cholinergic cells
in which the series resistance was <40 M were subjected to somatic
voltage clamp. Although voltage clamp under these circumstances can
produce distorted currents because of imperfect space clamp (Mainen et
al., 1995; White et al., 1995), we only wanted to determine whether a
sodium current was available in the subthreshold voltage range and did
not attempt to investigate kinetic properties. Under control
conditions, stepping from a clamp potential of 60 to 52.5 mV
produced an inward current that persisted throughout a 1 sec pulse
(Fig. 2C). The inward current was blocked by TTX
application, and repeating the same voltage step then revealed a small
outward current (Fig. 2D). Additionally, after TTX, a
subthreshold zero current point appeared at 60 mV (Fig.
2D). The current-voltage (I-V)
curve displayed a region of negative slope conductance between 60 and
50 mV that was TTX-sensitive (Fig. 2E).
Furthermore, voltage steps to 60 mV and above evoked a net outward
current in the presence of TTX (n = 9), indicating that
whatever additional inward currents are available in this portion of
the subthreshold voltage range are insufficient to drive firing in the
absence of the sodium current. The control and TTX data were subtracted
and pooled (n = 9) (Fig. 2F). These
data indicate that sodium channel activation occurs at potentials more
positive than approximately 60 mV, producing an inward current at
subthreshold membrane potentials that assists in driving tonic
firing.
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Figure 2.
The role of sodium in spontaneous tonic firing.
A, Spontaneous firing was observed in the majority of
cholinergic interneurons. B, Application of TTX (1 µM) prevented action potential generation and established
a stable subthreshold membrane potential (approximately 60 mV). C,
Stepping the membrane from a holding potential of 60 to 52.5 mV
elicited a slowly developing inward current that persisted throughout
the pulse. The dashed line indicates the zero current
point. D, After TTX (1 µM) treatment, the
inward current produced by the depolarizing voltage step was absent,
and a small outward current was observed. Additionally, the zero
current point was shifted to 60 mV. E, The I-V plot
for a range of voltage steps (Vcmd)
shows that, under control conditions, there is a region of negative
slope conductance at potentials positive to 60 mV and no zero current
point in the subthreshold voltage range. The inward current generated
by depolarizing steps was blocked by TTX. F, Examination
of the TTX-sensitive current that was obtained by subtraction for nine
neurons and pooled (mean ± SD). G-J, Spiking rate
was reduced by injection of constant negative current and failed to
reveal any subthreshold oscillation in the absence of action potential
generation. The firing pattern was related to firing rate, and spike
trains became increasingly irregular at lower rates. K,
For seven neurons exhibiting tonic, regular spiking (2.45-4.03 Hz; CV,
0.10-0.19), the firing rate and pattern was measured for control (0 pA) and during steady injection of 10 and 20 pA (open
circles). Pooled data (filled circles;
mean ± SD) confirmed that firing pattern was a function of
spiking rate.
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In certain types of neurons, spontaneous rhythmic firing has been shown
to arise from subthreshold oscillations that are action potential-independent (Llinas and Yarom, 1986; Shepard and Bunney, 1991; Kang and Kitai, 1993). Such oscillations can be revealed by
applying hyperpolarizing constant current that is just sufficient to
prevent action potential generation. We tested whether such subthreshold oscillations might underlie tonic firing in cholinergic cells by applying small-amplitude hyperpolarizing constant currents. A
reduction in the firing rate was seen but no subthreshold oscillations were revealed (Fig. 2G-J), although the membrane
potential traversed the same voltage range as it did under control
conditions. This suggests that the rhythmic fluctuation in the membrane
is not produced by an action potential-independent subthreshold
oscillation. Furthermore, it was found that, as the firing rate
decreased, the irregularity of the spike train increased. A group of
cholinergic cells (n = 7), which all exhibited
spontaneous regular firing (rate, 2.45-4.03 Hz; CV, 0.10-0.19) were
subjected to DC hyperpolarizing current injection, and examination of
the resulting spike trains confirmed that the CV was a function of the
firing rate (Fig. 2K). These observations raise the
possibility that the stochastic properties of the channels underlying
the subthreshold membrane potential fluctuations during spontaneous
firing contribute to the irregularity of cholinergic cell spike trains
(White et al., 2000).
Ih
Although the subthreshold sodium current provides an explanation
for how cholinergic neurons reach action potential threshold from
potentials of 60 mV and above, it does not account for the recovery
from the AHP. This suggests that at least one other mechanism must
provide a depolarizing influence at potentials more negative than 60
mV. Because cholinergic cells possess the hyperpolarization-activated cation current, Ih (Jiang and North,
1991; Kawaguchi, 1993), and this current is known to be involved in
pacemaker activity in many cell types (DiFrancesco, 1993; Maccaferri
and McBain, 1996; Pape, 1996; Lüthi and McCormick, 1998b), we
examined whether Ih provides a
depolarizing influence in the subthreshold voltage range. Blockade of
Ih with cesium (3 mM) produced a significant reduction in firing
rate (n = 9) (Fig.
3A-C, Table
1) and hyperpolarized the membrane
potential (n = 13) (Table 1). Confirmation that cesium
application blocked Ih was provided by
examining the response to negative current injection. Under control
conditions, there is an initial hyperpolarization followed by a sag in
the membrane potential as Ih becomes
activated (Fig. 3D). After cesium application, the sag is
absent, indicating that Ih is blocked
(Fig. 3D). Using somatic voltage clamp, we found that cesium
application reduced a slowly developing hyperpolarization-activated
inward current and produced a shift in the holding current at a clamp
potential of 60 mV (Fig.
3E,F). Examination of the
I-V curve under control conditions and after cesium
treatment shows that blocking Ih
established a zero current point at approximately 65 mV in this
neuron (Fig. 3G). Subtraction of the cesium-sensitive
current was used to allow examination of pooled data (n = 5), which revealed that Ih is activated and provides an inward current throughout the subthreshold voltage range observed in cholinergic cells (Fig.
3H). The h-current between 65 and 55 mV is
relatively small, which is consistent with this voltage range being at
the foot of the activation curve for
Ih (Hagiwara and Irisawa 1989;
Lüthi and McCormick 1998a, 1999). Because the voltage range over
which Ih and the subthreshold sodium
current overlap, the most likely explanation for spontaneous firing in
cholinergic interneurons is that these two currents together ensure
that there is no zero current point at any subthreshold membrane
potential. Although Ih is critical in
the subthreshold voltage range, examination of frequency-current
(f-I) and instantaneous f-I plots
revealed that the h-current did not contribute significantly to the
suprathreshold properties of cholinergic cells (data not shown;
n = 9).
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Figure 3.
Ih is required for
spontaneous firing. A, Tonic firing observed under
control conditions. B, Application of cesium (3 mM) produced a profound decrease in the firing rate and an
accompanying increase in the irregularity of the spike train.
C, After washout of cesium, the firing rate and pattern
were restored to control values. D, Injection of
negative current produced an initial hyperpolarization followed by a
sag in the membrane potential caused by activation of
Ih. Cesium abolished the sag, confirming
that Ih was blocked. E,
Stepping from a holding potential of 60 to 80 mV evoked a slowly
developing inward current. F, Application of cesium
reduced the amplitude of the inward current measured at the end of the
pulse and shifted the zero current point. G, The
I-V plot for a range of voltage steps
(Vcmd) from a holding potential of
60 mV reveals that cesium blocks an inward current that is activated
throughout the subthreshold voltage range. Cesium also established a
zero current point at approximately 65 mV in this neuron.
H, The cesium-sensitive current was obtained by
subtraction (n = 5) and exhibited the voltage
dependence expected for Ih.
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Calcium imaging
To investigate the role of calcium in tonic firing, we first used
imaging to determine whether fluctuations in calcium levels were
detectable in cholinergic cells. Conventional whole-cell recording
methods were used for this part of the study, and the neurons were
loaded with the calcium indicator fura-2 (50-200 µM) to
examine the intracellular calcium dynamics. In response to
small-amplitude (20-80 pA) depolarizing current injections (1-5 sec),
cholinergic cells generated trains of spikes with each spike associated
with an elevation in intracellular calcium (n = 28),
which was detectable in both somatic and dendritic compartments (Fig.
4A,B).
Hyperpolarization from subthreshold membrane potentials (50 to 80
mV; n = 10) failed to produce any detectable reduction in intracellular calcium (Fig. 4C), suggesting that calcium
entry was primarily spike-triggered. Furthermore, the rebound in the membrane potential that followed hyperpolarization also failed to
generate a detectable elevation in calcium level (Fig. 4C). However, there remains the possibility that a T-type calcium current was contributing to the rebound but produced an insufficient elevation in the proximal intracellular calcium concentration to be detected optically. We therefore examined the rebound after hyperpolarizing current injection (500 msec, 200 pA) in nine neurons under control conditions and after the application of TTX (1 µM) and cesium (3 mM;
n = 4) or ZD7288 (100 µM;
n = 5) to block Ih.
Blockade of sodium channels reduced the amplitude of the rebound,
indicating that a sodium current contributed to bringing the neuron to
threshold (Fig. 4D). After blockade of
Ih, no time-dependent sag in the membrane potential was observed in response to the hyperpolarizing current pulse and the rebound was completely absent (Fig.
4E). Identical observations were made for nine
neurons and indicate that, if a T-type calcium current is present, it
is of insufficient magnitude to account for the rebound. These data
also suggest that a T-current is unlikely to play a significant role in
the depolarizing ramp to spike threshold during spontaneous tonic firing.
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Figure 4.
Voltage dependence of calcium influx.
A, Fura-2-loaded cholinergic neuron at an excitation
wavelength of 380 nm. The red box over the soma and the
blue boxes over the dendrites are the areas from which
the fluorescence signals were measured. B, Examination
of the response to current injection (+75 pA) revealed that each spike
was associated with a calcium elevation in both the soma and dendrites.
C, Injection of negative current (75 pA) produced a
hyperpolarization and a subsequent sag in the membrane potential
attributable to activation of Ih. After
termination of the current pulse, a rebound in the membrane potential
was observed. There was no detectable alteration in the fluorescence
signal either in response to the hyperpolarization or the rebound.
Calcium signals illustrated B and C are
individual sweeps. D, Under control conditions,
injection of negative current (200 pA) produced a hyperpolarization
and a subsequent sag in the membrane potential, which was followed by a
rebound in the membrane potential after termination of the pulse.
Application of TTX (1 µM) reduced the amplitude the
rebound. E, Blockade of Ih
eliminated the sag during membrane hyperpolarization and abolished the
rebound.
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Spontaneous firing was also observed in fura-loaded cholinergic cells,
albeit less frequently than with perforated patch recording because of
the previously described effects of intracellular dialysis (Bennett and
Wilson, 1999). In neurons that continued to fire spontaneously
(n = 10), the spike-associated elevations in
intracellular calcium were very similar to those produced by current
injection (Fig.
5A,B).
The relationship between the membrane potential and the time at which
calcium elevations were observed was determined by examining data
collected during spontaneous firing after the neuron had reached steady
state. Using the maximal available temporal resolution, it was found
that very little elevation in calcium occurred during the depolarizing
ramp to spike threshold and that increases in the intracellular calcium
concentration were primarily spike-triggered (n = 10)
(Fig. 5C). Because sodium channel blockade established a
resting membrane potential that was ~ 11 mV below action
potential threshold and no net inward current was evoked by voltage
steps to 60 mV and above in the presence of TTX (see above), we
propose that subthreshold calcium entry plays a minor role in driving
tonic firing.
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Figure 5.
Calcium dynamics during spontaneous firing.
A, Fluorescence image (380 nm excitation) of a
cholinergic neuron loaded with fura-2 during conventional whole-cell
recording. The colored boxes are the locations from
which the fluorescence measurements were obtained. B,
Simultaneous fluorescence and electrical measurements during
spontaneous firing revealed concurrent spike-triggered elevations in
calcium concentration in the soma and primary dendrite
(bracket indicates data shown in C).
C, Examination of a single spike and the associated
calcium transient during spontaneous firing confirms that elevations in
intracellular calcium are primarily spike-triggered with minimal
calcium influx occurring during the depolarizing ramp to action
potential threshold. The dashed lines demarcate the
individual frames (30 msec) for the fluorescence data. Calcium signals
illustrated B and C are individual
sweeps.
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Calcium
The role of calcium in spontaneous tonic firing was investigated
by blocking calcium channels by either substituting equimolar cobalt (2 mM) for calcium or adding cadmium (400 µM) to
the ACSF. Cobalt or cadmium treatment silenced the majority (10 of 14)
neurons tested and resulted in the emergence of burst firing in the
remaining four cells (Fig.
6A,B).
In the other 10 cholinergic neurons, steady depolarizing current
(10-60 pA) was used to reinstate spiking and elicited clustered or
burst firing (data not shown). Measurement of the mean subthreshold
membrane potential revealed that calcium channel blockade produced a
significant hyperpolarization of the membrane potential
(n = 20) (Table 1). This was somewhat surprising in
light of the fact that the imaging data, voltage-clamp recordings, and
the current-clamp investigation of the rebound response all suggest
that subthreshold calcium currents are insufficient to drive
cholinergic neurons to spike threshold. The observation that
depolarizing current restored spiking suggested that preventing calcium
entry had reduced the availability of a subthreshold inward current. A
possible explanation is that blocking calcium channels produces a
hyperpolarizing shift in voltage range of activation of
Ih by reducing the intracellular
calcium concentration (Hagiwara and Irisawa, 1989; Lüthi and
McCormick, 1998a) or via calcium-dependent regulation of intracellular
cAMP (DiFrancesco and Tortora, 1991; DiFrancesco and Mangoni, 1994;
Lüthi and McCormick, 1999). Using somatic voltage clamp, we found
that application of cadmium or cobalt caused a reduction in the current
that was elicited with hyperpolarizing voltage steps (Fig.
6C,D). The I-V curve generated before
and after calcium channel blockade (Fig. 6E) shows
that cadmium reduced an inward current that was available throughout the subthreshold voltage range. The current blocked by cadmium or
cobalt was obtained by subtraction, and the data were pooled (n = 5). A large cell to cell variability was observed,
which is apparent from the SD (Fig. 6F), and although
a clear voltage-dependence was not observed, the subtracted current was
similar to the h-current in that it was activated throughout the
subthreshold voltage range (compare Figs. 3H,
6F). We therefore propose that the hyperpolarization that occurs as a consequence of blocking calcium channels arises primarily from a reduction in the availability of
Ih, which is produced by a direct or
indirect calcium-dependent hyperpolarizing shift in the voltage range
of activation of this current. However, we cannot rule out the
possibility that the hyperpolarization produced by calcium channel
blockade might also be contributed to by reduction of a subthreshold
calcium current.
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Figure 6.
Calcium is critical for tonic firing.
A, Spontaneous tonic firing. B,
Application of cadmium (400 µM) converted tonic spiking
to rhythmic bursting, during which clusters of spikes were generated in
rapid succession and separated by slow subthreshold oscillations in the
membrane potential. C, Stepping from a holding potential
of 60 to 80 mV activated the slowly developing h-current.
D, After calcium channel blockade, the same voltage step
produced a smaller current and was accompanied by a shift in the
holding current at 60 mV. E, The I-V
plot for this neuron generated using a range of voltage steps
(Vcmd) indicates that there has been
a reduction in a subthreshold inward current. F, The
current that was reduced by calcium channel blockade was obtained by
subtraction and pooled (n = 5;
symbols indicate mean ± SD) G,
Under control conditions, cholinergic neurons generate relatively broad
spikes that exhibit a shoulder on the falling phase. Calcium channel
blockade produced a pronounced narrowing of the spike and abolished the
shoulder. H, Application of cadmium abolished the slow
AHP that normally follows individual spikes.
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In addition to causing hyperpolarization, blockade of calcium channels
also produced profound alterations in the spiking properties of
cholinergic cells. Under control conditions, the action potential waveform exhibits a shoulder during the repolarizing phase (Fig. 6G) (see Fig. 8B). After calcium channel
blockade spike width was significantly reduced (n = 16)
(Table 1) and the shoulder was no longer visible (Fig. 6G),
indicating that calcium influx contributes to the time course and shape
of the action potential waveform. Examination of the AHP before and
after calcium channel blockade illustrates that the slow AHP that
normally follows individual spikes is calcium-dependent because it is
almost completely absent when calcium channels are blocked (Fig.
6H, Table 1) (Kawaguchi, 1992). The single-spike AHP,
which typically lasts a few hundred milliseconds, is one of the
determinants of the interspike interval and reducing calcium influx,
and therefore the amplitude of the AHP might be expected to alter the
response of cholinergic cells to depolarizing input. Examination of
firing elicited by positive current injection confirmed that calcium
plays a crucial role in regulating spike timing. Under control
conditions, small-amplitude (20-140 pA) depolarizing current
injections produced regular spike trains that exhibited some adaptation
at higher firing rates (Fig. 7A) (Kawaguchi, 1992). After
calcium channel blockade with cadmium or cobalt, cholinergic neurons
fired many more spikes in response to a given current pulse (Fig.
7B), which resulted in a profound increase in the slope of
the f-I relationship and a several-fold increase in the
instantaneous f-I relationship (Fig. 7C). The effects of cadmium and cobalt were indistinguishable, and the data were
therefore pooled (n = 13) and provided confirmation that calcium is critical for regulating the interspike interval (Fig.
7C,D). Examination of the instantaneous
f-I plots shows that, after the blockade of calcium
channels, there is considerable spike frequency adaptation during
high-frequency firing (Fig. 7C,D). Additionally,
there is a large, persistent hyperpolarization that lasts for >1 sec
after the episode of firing (Fig. 7B), raising the
possibility that both adaptation and the hyperpolarization result from
activation of a voltage-dependent outward current that accumulates
during the spike train. Cholinergic neurons are known to possess a
slowly activating voltage-dependent potassium current that could
underlie these phenomena (Song et al., 1998).
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Figure 7.
Calcium regulates the firing properties of
cholinergic neurons. A, In response to depolarizing
current injection (+40 pA; left), cholinergic cells
generated spikes that were separated by a slow AHP. Injection of larger
amplitude currents (+140 pA; right) evoked repetitive
spiking with minimal spike frequency adaptation. B,
After application of cadmium (400 µM), the same cell
generates many more spikes in response to a given current injection and
spike-frequency adaptation. C, Calcium channel blockade
produces a profound increase in the slope of the f-I
relationship (left). The instantaneous
f-I plot (+140 pA; right) shows a
pronounced increase in the initial firing rate and substantial spike
frequency adaptation after blockade of calcium channels.
D, Pooled data (n = 13, 9 cadmium, 4 cobalt; symbols indicate mean ± SD) confirm that
calcium channel blockade profoundly alters both the f-I
and instantaneous f-I relationships.
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Calcium: BK plus SK channels
Because calcium influx was clearly involved in shaping the action
potential waveform and in the generation of the AHP, we investigated
whether calcium-activated potassium currents might contribute to spike
repolarization and determined the identity of the current underlying
the AHP. BK channels are involved in the repolarization process in many
cell types. BK channels are readily blocked by low concentrations of
TEA, but there are several types of voltage-dependent potassium
channel, including the Kv3 family of currents, Kv1.1, and KCNQ2
(Coetzee et al., 1999; Rudy et al., 1999), which are also blocked by
low concentrations of TEA. We first determined whether TEA (1 mM) had any effects on the spike waveform after first
blocking calcium channels with cadmium or cobalt. In the presence of
calcium channel blockers, application of TEA was without effect
(n = 4) and produced no detectable alteration in spike
width or threshold (Fig.
8A), indicating that
voltage-dependent potassium channels sensitive to low-dose TEA do not
play an important role in spike repolarization in cholinergic cells.
However, when calcium influx was not attenuated, addition of TEA (1 mM; n = 5) produced significant
spike broadening (Table 1). This effect was mimicked by application of
iberiotoxin (100 nM; n = 2) (Fig.
8B), indicating that spike-triggered elevations of
intracellular calcium activate BK channels, which then contribute to
spike repolarization.
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Figure 8.
Spike-triggered calcium entry activates BK
channels, which contribute to repolarization. A,
Application of TEA (1 mM) after cadmium treatment had no
discernable effect on action potential width. B, In the
presence of calcium-containing ACSF, spike width was significantly
broadened after treatment with TEA or iberiotoxin (100 nM).
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The AHP that follows individual spikes in cholinergic cells is almost
completely lost after cadmium or cobalt treatment (Fig. 6E), suggesting the involvement of a calcium-mediated
process. This was confirmed using apamin (100 nM;
n = 6), which blocks a class of SK calcium-activated
potassium channels. The most obvious effect of blocking SK channels was
that spontaneous tonic firing was converted to rhythmic bursting (Fig.
9A,B).
Bursting was characterized by spike clusters separated by
large-amplitude oscillations of the membrane potential. The amplitude
of the AHP after individual spikes within a burst was significantly
reduced (Fig. 9C) and the spike was significantly broadened
(Table 1). Although SK channels are not thought to be involved in spike
repolarization directly, apamin might cause spike broadening by
blocking any SK channels that are open at subthreshold voltages. The
response to current injection was altered after apamin treatment (Fig. 9D,E), and the instantaneous firing
rate was found to be elevated at the beginning of the spike train (Fig.
9F). There was rapid adaptation, and frequently
firing ceased before termination of the current injection, which was
followed by a long-duration hyperpolarization. Spike cessation likely
resulted from accumulation of a voltage-activated outward current and
perhaps the activation of an apamin-insensitive calcium-activated
potassium current.
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Figure 9.
Calcium-activated SK channels underlie the AHP and
regulate spike patterning. A, Under control conditions,
spontaneous tonic firing was observed. B, Application of
apamin (100 nM), which blocks calcium-dependent SK
channels, converted tonic firing to rhythmic bursting.
C, The AHP after individual spikes was completely
blocked by apamin, but between bursts, there was a large slow
hyperpolarization. D, Injection of depolarizing current
elicited a train of spikes that displayed spike frequency adaptation.
E, After application of apamin, the same current pulse
evoked spiking with a higher initial firing rate but with much more
pronounced adaptation and resulted in spike cessation before
termination of the current pulse. F, Apamin produced an
elevation in the instantaneous firing rate and more pronounced
spike-frequency adaptation (n = 5;
symbols indicate mean ± SD).
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DISCUSSION |
Spontaneous tonic firing in neostriatal cholinergic interneurons
is generated by the intrinsic membrane properties, which ensure that
there is no zero current point in the subthreshold voltage range and
therefore no resting membrane potential. Thus, endogenous excitability
is central to spike timing in cholinergic cells. The spontaneous firing
rates and patterns observed in this study indicate that the intrinsic
membrane properties are probably primarily responsible for the
generation of the ongoing spike sequences, which are characteristic of
TANs recorded in vivo.
Sodium and Ih drive spiking
The combined depolarizing action of a subthreshold sodium current
and Ih drive cholinergic cells to
spike threshold. The voltage range over which these two currents are
activated overlap, producing an inward current at all subthreshold
membrane potentials. Blockade of sodium channels with TTX established a
stable resting membrane potential, which was ~10 mV negative to
spike threshold, indicating that the sodium current is required to
bring cholinergic cells to a voltage at which spikes can be initiated.
Somatic voltage clamp revealed the presence of a TTX-sensitive inward
current that was activated at membrane potentials of approximately 60 mV and above. In the presence of TTX, the region of negative slope conductance between 60 mV and threshold was absent, and a
subthreshold zero current point was established. Action
potential-independent oscillations in the membrane potential
were not observed, and application of hyperpolarizing current failed to
reveal any subthreshold oscillation that might participate in the
generation of tonic activity. Together, these data indicate that the
subthreshold sodium current is critically involved in driving tonic
firing. A subthreshold or persistent sodium current has been described in many classes of neurons (Crill, 1996), including neostriatal cholinergic cells (Chao and Alzheimer, 1995), and is sufficient to
drive tonic activity in cerebellar Purkinje cells, subthalamic neurons,
dopaminergic retinal amacrines, and neurons of the suprachiasmatic nucleus (Pennartz et al., 1997; Feigenspan et al., 1998; Bevan and
Wilson, 1999; Raman and Bean, 1999). However, in cholinergic neurons,
the spike AHP hyperpolarizes the neuron to a voltage range at which
there is minimal sodium current activation, suggesting that at least
one other subthreshold inward current is required for tonic firing.
Cholinergic cells possess the hyperpolarization-activated mixed cation
conductance, Ih (Jiang and North,
1991; Kawaguchi, 1992, 1993). In many types of neurons and in heart,
Ih is described as the pacemaker
current because it provides a depolarizing influence and regulates the
time course of rhythmic oscillatory activity (DiFrancesco, 1993;
Maccaferri and McBain, 1996; Pape, 1996; Lüthi and McCormick,
1998b). Blockade of Ih in cholinergic
neurons produced a profound reduction of firing rate and, in four of
nine, neurons resulted in complete quiescence. Voltage-clamp data
revealed that Ih was activated at all
subthreshold voltages and that blockade of this current could give rise
to a zero current point in the subthreshold range and therefore a
stable resting membrane potential. The h-current was required for
recovery from the AHP and provided a sufficient depolarizing influence
to drive the membrane potential into the voltage range at which the
sodium current became available. Although
Ih was activated at all subthreshold
voltages, it appeared to be insufficient to bring cholinergic cells to
spike threshold in the absence of the subthreshold sodium current (see
above). Thus, one possible explanation for the range of firing rates
observed for cholinergic cells in vitro and the fact that
some cholinergic cells do not exhibit spontaneous activity is that
there is cell to cell variability in the magnitude of
Ih and the sodium current, both of
which are required to ensure that there is no zero current point in the
subthreshold voltage range.
Calcium-dependent potassium currents pace tonic firing
The temporal and spatial calcium dynamics were investigated for
both spontaneous firing and during spiking elicited by intracellular current injection using fluorescence microscopy and simultaneous electrical recording. Minimal alterations in fluorescence were observed
throughout the subthreshold voltage range, including the depolarizing
ramp to spike threshold, and almost all elevations in intracellular
calcium appeared to be spike-triggered. Cholinergic cells do not seem
to possess a significant T-type calcium current because elevations in
intracellular calcium were not seen during rebound depolarizations
after negative current injection and rebounds were altogether absent
after blockade of the sodium current and Ih. Spike-triggered calcium influx was
therefore likely to be mediated by high voltage-activated calcium
channels that appeared to be located in both the soma and dendrites
because action potentials elicited simultaneous elevations in calcium
in both locations. Calcium currents are therefore likely to play a
secondary role in directly driving tonic firing but appear to be of
primary importance in the maintenance of spontaneous activity by virtue
of the apparent calcium-dependence of
Ih. The voltage range of activation of
Ih can be shifted directly by calcium
(Hagiwara and Irisawa, 1989; Lüthi and McCormick, 1998a) or
indirectly through calcium-dependent regulation of intracellular cAMP
(DiFrancesco and Tortora, 1991; DiFrancesco and Mangoni, 1994;
Lüthi and McCormick, 1999). Because minimal calcium entry appears
to occur at subthreshold membrane potentials, we propose that blocking
calcium channels hyperpolarizes cholinergic cells primarily by reducing
the availability of Ih. In addition,
it is possible that the hyperpolarization may have also been
contributed to by reduction of a subthreshold calcium current that was
activated over the same voltage range as
Ih.
Calcium influx is pivotal in shaping the action potential and in the
subsequent activation of both BK and SK type calcium-dependent potassium channels, which are involved in spike repolarization and in
the generation of the AHP, respectively. The action potential waveform
exhibits a characteristic shoulder on the falling phase that is similar
to that produced by the calcium component of the spike in inferior
olivary neurons (Llinas and Yarom, 1981a,b). After calcium channel
blockade, the spike width was reduced by 40% and the shoulder was
absent, confirming the role of calcium in regulating the action
potential time course and shape. The fact that rapid repolarization
occurs in the absence of calcium-dependent outward currents indicates
that voltage-dependent potassium currents are activated during spiking.
However, the Kv3 family of currents, Kv1.1, and KCNQ2 do not appear to
be involved in spike repolarization because 1 mM TEA did
not alter spike width during calcium channel blockade. Cholinergic
neurons possess a well characterized A-type potassium current (Song et
al., 1998), which is likely to play a role in the repolarization
process. Under control conditions, spike-triggered calcium influx
activated BK channels, which assisted in repolarization as they do in a
diversity of neurons (Adams et al., 1982; Lancaster and Nicoll, 1987;
Brown et al., 1990; Storm, 1990; Sah and McLachlan, 1992; Sah, 1996).
Thus, both calcium-dependent and -independent outward currents appear
to be required for spike termination and repolarization. In addition,
spike-triggered calcium entry activates apamin-sensitive SK channels
that underlie the single-spike AHP. The AHP is similar in
pharmacological profile and duration to the medium AHP that has been
described in many different types of cells (Pennefather et al., 1985;
Schwindt et al., 1988; Shepard and Bunney, 1991; Sah and McLachlan,
1992; Kohler et al., 1996; Ping and Shepard, 1996; Sah, 1996; Bond et al., 1999). After calcium channel blockade and reduction of the AHP,
there is a transformation in the firing properties of cholinergic cells
with a threefold to fourfold increase in mean firing rate produced by
current injections of 60-140 pA. The time course of the AHP,
particularly the rate of deactivation, is therefore critical in
determining the interspike interval. Furthermore, both direct calcium
channel blockade or application of apamin, which blocks SK channels,
could convert tonic firing to rhythmic bursting, indicating that the
AHP is of pivotal importance in regulating the spontaneous firing pattern.
The generation of tonic firing
The intrinsic membrane properties of cholinergic cells provide an
explanation for the origin of spontaneous tonic firing in cholinergic
neurons. A TTX-sensitive fast sodium current generates the spike that
activates calcium channels, and the resulting calcium influx shapes the
action potential. Both voltage-dependent (probably A-type) and
calcium-dependent (BK) potassium channels assist in repolarization, and
spike-triggered calcium entry also activates SK channels, which produce
the slow AHP. The AHP brings the neuron into a voltage range at which
Ih produces a sufficient inward current to cause depolarization as the AHP decays. Finally,
Ih depolarizes the membrane potential
to a point at which the subthreshold sodium current is activated, the
cell is driven to threshold, and the cycle is repeated. A possible
explanation for burst firing is that reducing the amplitude of the AHP
results in insufficient repolarization for the neuron to escape the
voltage range of activation of the subthreshold sodium current.
Consequently, a rapid succession of spikes are generated, and the burst
is terminated by either calcium-dependent apamin-insensitive potassium
channels and/or accumulation of a slowly activating noninactivating
potassium current, the latter of which has been described for
cholinergic cells (Song et al., 1998). Recovery from the interburst
hyperpolarization would arise from activation of
Ih and decay of the mechanism(s) responsible for the hyperpolarization.
Implications for spike timing in TANs
Unit recordings from awake, behaving monkeys have described a
spectrum of firing patterns for TANs ranging from tonic regular or
irregular firing to bursting. A very similar range of firing patterns
is observed for cholinergic cells in vitro. Although synaptic inputs are clearly influential in regulating spike timing in
cholinergic cells, we propose that the characteristic spike sequences
described for TANs recorded in vivo are a natural product of
the intrinsic membrane properties. Furthermore, we suggest that
bursting may arise from neuromodulatory regulation of calcium influx,
which controls the AHP, and that the persistent oscillatory activity of
TANs detected in parkinsonian states could reflect a pathophysiological
switch to burst firing mode.
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FOOTNOTES |
Received May 24, 2000; revised Sept. 1, 2000; accepted Sept. 5, 2000.
This study was supported by National Institutes of Health Grant
NS37760. B.D.B. thanks Profs. S. T. Kitai and W. E. Armstrong for providing accommodation at The University of Tennessee, Memphis during January through March, 2000.
Correspondence should be addressed to Charles J. Wilson, Cajal
Neuroscience Research Center, Division of Life Sciences, University of
Texas, San Antonio, 6900 North Loop, 1604 West, San Antonio, TX 78249. E-mail: cjwilson{at}utsa.edu.
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TOP
ABSTRACT
INTRODUCTION
