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The Journal of Neuroscience, March 1, 1999, 19(5):1663-1674
Ionic Currents Underlying Spontaneous Action Potentials in
Isolated Cerebellar Purkinje Neurons
Indira M.
Raman and
Bruce P.
Bean
Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts 02115
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ABSTRACT |
Acutely dissociated cell bodies of mouse Purkinje neurons
spontaneously fired action potentials at ~50 Hz (25°C). To directly measure the ionic currents underlying spontaneous activity, we voltage-clamped the cells using prerecorded spontaneous action potentials (spike trains) as voltage commands and used ionic
substitution and selective blockers to isolate individual currents. The
largest current flowing during the interspike interval was
tetrodotoxin-sensitive sodium current (approximately 
50 pA between
65 and 60 mV). Although the neurons had large voltage-dependent
calcium currents, the net current blocked by cobalt substitution for
calcium was outward at all times during spike trains. Thus, the
electrical effect of calcium current is apparently dominated by rapidly
activated calcium-dependent potassium currents. Under current clamp,
all cells continued firing spontaneously (though ~30% more slowly) after block of T-type calcium current by mibefradil, and most cells
continued to fire after block of all calcium current by cobalt
substitution. Although the neurons possessed
hyperpolarization-activated cation current
(Ih), little current flowed during
spike trains, and block by 1 mM cesium had no effect on
firing frequency. The outward potassium currents underlying the
repolarization of the spikes were completely blocked by 1 mM TEA. These currents deactivated quickly (<1 msec) after
each spike. We conclude that the spontaneous firing of Purkinje neuron
cell bodies depends mainly on tetrodotoxin-sensitive sodium current
flowing between spikes. The high firing rate is promoted by large
potassium currents that repolarize the cell rapidly and deactivate
quickly, thus preventing strong hyperpolarization and restoring a high
input resistance for subsequent depolarization.
Key words:
cerebellum; sodium current; calcium current; potassium
current; Ih; spike; pacemaking; pacemaker
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INTRODUCTION |
Brain activity must begin somewhere,
and some neurons spontaneously fire action potentials even in the
absence of external stimuli. Such pacemaking activity is seen in a wide
variety of neurons in the CNS (Llinás, 1988
), including thalamic
neurons (Jahnsen and Llinás, 1984; McCormick and Pape, 1990),
neurons of the suprachiasmatic nucleus (Pennartz et al., 1997), neurons releasing monoamine transmitters such as dopamine, serotonin, histamine, and norepinephrine (Williams et al., 1984; Grace and Onn,
1989; Yung et al., 1991; Uteshev et al., 1995; Bayliss et al., 1997),
and GABAergic interneurons of the hippocampus and the cerebral and
cerebellar cortex (Alger and Nicoll, 1980; Salin and Prince, 1996;
Häusser and Clark, 1997).
The ionic mechanisms underlying spontaneous firing are incompletely
understood. In pacemaker cells of the heart, a
hyperpolarization-activated cation current
(Ih) is important for spontaneous
activity (DiFrancesco, 1993). This current also contributes to the
spontaneous activity of some central neurons (Pape, 1996).
Low-threshold, T-type calcium channels also contribute to the
spontaneous activity of some excitable cells, including sinoatrial
cells, neuroendocrine cells, and various central neurons (Huguenard,
1996). Of central neurons that fire spontaneously, the most thorough
investigation has been made of thalamic neurons, where both
Ih and T-type calcium channels play important
roles (Jahnsen and Llinás, 1984; McCormick and Pape, 1990).
However, in other neurons, spontaneous firing does not require
Ih or calcium current. In some,
tetrodotoxin-sensitive sodium current appears to be important in
generating spontaneous depolarizations as well as in forming spikes
(Alonso and Llinás, 1989; Pennartz et al., 1997; Feigenspan et
al., 1998).
Cerebellar Purkinje neurons in vivo show regular,
spontaneous firing (Bell and Grimm, 1969; Latham and Paul, 1971).
Studies with in vitro preparations suggest that this results
from intrinsic membrane properties. Spontaneous firing can be
maintained in cerebellar slice preparations (Hounsgaard, 1979;
Llinás and Sugimori, 1980b; Häusser and Clark, 1997) and in
cultured Purkinje neurons (Gruol and Franklin, 1987), even when
synaptic activity is blocked. Early studies suggested that spontaneous
firing was caused by calcium action potentials arising in the dendritic
tree (Llinás and Sugimori, 1980b). Surprisingly, however,
cultured Purkinje neurons were found to fire spontaneously even before
formation of dendrites (Gruol et al., 1991), and subsequent work has
shown that spontaneous firing is preserved even in acutely isolated
cell bodies of Purkinje neurons (Nam and Hockberger, 1997; Raman and
Bean, 1997).
Most efforts to understand how different ionic conductances
interact to produce spontaneous activity in central neurons have relied
on computer models. Here we have taken advantage of the ability to
obtain high-quality voltage-clamp recordings from isolated Purkinje
neurons to take a direct experimental approach. By using previously
recorded spontaneous action potentials as voltage commands, we measured
the ionic currents that flow during spontaneous activity, using
pharmacology and ionic substitution to identify components of current
from particular channel types.
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MATERIALS AND METHODS |
Cell preparation. Experiments were performed on
cerebellar Purkinje neurons enzymatically isolated as previously
described (Mintz et al., 1992; Raman et al., 1997). Black Swiss mice
(postnatal day 14-20) were anesthetized with methoxyflurane before
decapitation, and the vermal layer of the cerebellum was removed and
minced in ice-cold, oxygenated dissociation solution containing (in
mM): 82 Na2SO4, 30 K2SO4, 5 MgCl2, 10 HEPES, 10 glucose, and 0.001% phenol red (buffered to pH 7.4 with
NaOH). The tissue was then incubated for 7 min in 10 ml of dissociation
solution containing 3 mg/ml protease XXIII (Sigma, St. Louis, MO), pH
7.4 with NaOH, at 37°C, with oxygen blown over the surface of the
fluid. The tissue was then washed in warmed, oxygenated dissociation
solution containing 1 mg/ml bovine serum albumin and 1 mg/ml trypsin
inhibitor, and then maintained in Tyrode's solution containing (in
mM): 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 glucose, pH 7.4 with NaOH, at
room temperature, with oxygen blown over the surface of the fluid.
Tissue was withdrawn as needed and triturated with a fire-polished
Pasteur pipette to liberate individual neurons. Purkinje cells were
identified by their large diameter and characteristic pear shape
attributable to the stump of the apical dendrite. Cells were used
between 30 min and 5 hr of trituration. Although long dendrites were
rarely seen, some dendritic membrane may have been incorporated into
the isolated neurons.
Current-clamp experiments. Recordings were made with an
Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Borosilicate pipettes (3-5 M
) were filled with an internal solution containing (in mM): 122 KCH3O3S, 9 EGTA, 9 HEPES, 1.8 MgCl2, 14 Tris-creatinePO4, 4 MgATP, and 0.3 TrisGTP, buffered
to pH 7.4 with KOH. This solution was designed to approximate
physiological ionic conditions. The calcium-buffering properties are
likely to differ from endogenous conditions (Fierro and Llano, 1996),
but it seems unlikely that this would greatly affect firing properties
because even complete block of calcium entry generally had small
effects. The control external solution was Tyrode's solution. Voltage
values were corrected for a 5 mV junction potential between these
solutions. Spontaneous action potentials were recorded in the absence
of injected current. For the voltage-clamp/current-clamp switch for the
experiments shown in Figures 13 and 14, cells were voltage-clamped in
the single-electrode continuous voltage-clamp mode for 4-6 sec
(depending on the protocol) and then the clamp was released manually by
switching into bridge mode.
Voltage-clamp experiments. Whole-cell recordings were made
at room temperature with an Axopatch 200B amplifier (Axon Instruments). For all experiments except those of Figure 8 (calcium currents), borosilicate pipettes (1-3 M) were filled with the same internal solution as for the current-clamp experiments. For the experiments of
Figure 8, pipettes contained (in mM): 117 CsCl, 9 EGTA, 9 HEPES, 1.8 MgCl2, 14 Tris-creatinePO4, 4 MgATP, and 0.3 TrisGTP, buffered to pH 7.4 with CsOH.
Capacitative current was removed using the capacity compensation
circuitry of the patch-clamp amplifier. Sometimes capacity compensation
was imperfect, as indicated by small transients at the beginning of
step voltage pulses. Series resistance was compensated by 85-95%.
Solution changes. Cells were lifted and placed in front of a
row of flow pipes. The control external solution was normal Tyrode's solution, referred to as "calcium Tyrode's solution." Other
solutions included "cobalt Tyrode's solution" in which 2 mM CoCl2 was substituted for the 2 mM CaCl2, and TTX, TEA, and Cs in which
300 nM TTX (except where lower concentrations are
indicated), 1 mM tetraethylammonium chloride, or 1 mM CsCl, respectively, was added to the calcium or cobalt
Tyrode's solution. For isolation of sodium, potassium, calcium, and
Ih currents, protocols were repeated in
Tyrode's solution plus the relevant blocker, and these recordings were subtracted from the control records. Although raw traces are
illustrated in some of the figures, quantitative measurements were made
only on subtracted records, which isolate the ionic current of interest.
All experiments were performed at room temperature (20-24°C)
Drugs. Mibefradil and nimodipine were kindly provided by Dr.
Eric Ertel (Hoffmann-La Roche, Basel, Switzerland) and Dr. Richard McCarthy (Miles Laboratories, West Haven, CT), respectively. All other
chemicals were obtained from Sigma, except 
-conotoxin-GVIA (Peninsula Laboratories, Belmont, CA), -conotoxin-MVIIC (Bachem California, Torrance, CA), and -agatoxin-IVA (Peptides
International, Louisville, KY).
Acquisition and analysis. Data were acquired with a Digidata
1200 interface and pCLAMP software (Axon Instruments) and analyzed with
pClamp and Origin (Microcal Inc., Northampton, MA). Data are presented
as mean ± SEM.
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RESULTS |
Firing properties of isolated Purkinje neurons
All isolated Purkinje neurons fired spontaneously (with no
injected current) when studied in current clamp with physiological solutions. Each cell fired at an extremely regular rate, with frequencies across cells ranging from 17 to 148 Hz. The neuron whose
action potentials are illustrated in Figure
1A fired at 47.5 Hz,
with interspike intervals over 182 action potentials varying by only a
few milliseconds (Fig. 1B). In 34 cells, the median
firing frequency was 47 Hz, and the mean was 54 ± 5 Hz (Fig.
1C). The most negative voltage reached between action
potentials was 71 ± 1 mV, and the peak of the spikes was
+11 ± 2 mV.
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Figure 1.
Properties of spontaneous firing in isolated
Purkinje neurons. A, Spontaneous action potentials
recorded from a Purkinje neuron bathed in physiological saline. In this
and all current-clamp traces (except voltage-current relations),
dotted lines indicate 0 and 70 mV. This record of
spontaneous firing was used as the spike train command potential for
voltage-clamp experiments. B, Interspike interval
histogram for the cell in A. The mean ± SD was
20.5 ± 1.1 msec (182 intervals), giving a coefficient of
variation of 0.05. C, Histogram of spontaneous firing
frequencies of 34 Purkinje neurons.
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Voltage-clamp experiments: sodium currents
We studied the neurons under voltage-clamp to characterize the
ionic currents underlying spontaneous firing. In voltage-clamped neurons, currents were elicited both by step depolarizations and by
using a previously recorded train of spontaneous action potentials ("spike train") as the command voltage-clamp waveform. We
identified particular ionic currents present in the cells by
conventional measurements of voltage dependence and kinetics during the
step depolarizations, together with ionic substitution and blocking agents to isolate individual components of current. The spike train
protocol enabled us to make direct measurements of pharmacologically isolated currents flowing at each point in the cycle of pacemaking.
In principle, the record of a cell's own spontaneous firing could be
used as the command voltage for that cell. Then the total current
(ionic current plus capacitative current) flowing in response to the
command voltage should be zero at all times during the cycle, with
ionic current and capacitative current equal and opposite. This
experiment has been accomplished in cardiac sinoatrial cells by
switching a patch-clamp amplifier between current-clamp and voltage-clamp modes (Doerr et al., 1989; Zaza et al., 1997). This was
not possible in Purkinje neurons, where action potentials are faster
and currents are bigger. We found that the voltage recorded by the
patch-clamp amplifier in current-clamp mode was significantly distorted
(with an artifactually negative trough potential and artifactually
positive spike peak) compared with that recorded using an amplifier
with a voltage-follower head stage. The reasons for the distortion of
current-clamp recordings using a patch-clamp amplifier have been
discussed (Magistretti et al., 1996).
In making voltage-clamp recordings, we therefore used as command
voltage a record of spontaneous firing previously recorded in a
different cell using a voltage follower amplifier. We selected a record
of spontaneous firing from a cell that had a firing frequency near the
median value across cells. Because we were interested in ionic currents
flowing during the spike train, we removed the capacitative current
using the capacity compensation circuitry of the patch-clamp amplifier.
We then used selective blocking agents to isolate individual components
of ionic current.
In identifying individual currents active during cycles of pacemaking,
we first focused on voltage-dependent sodium current. All
voltage-dependent sodium current in Purkinje neurons is blocked by TTX
(Song and Narahashi, 1996; Nam and Hockberger, 1997; Raman and Bean,
1997; Kay et al., 1998), and the high selectivity and complete
reversibility of TTX make it an ideal tool for isolating a single
component of current elicited in response to a complex waveform. Figure
2 shows currents evoked by step
depolarizations from a holding potential of 60 mV
(A) and by the spike train protocol
(B). These voltage-clamp experiments used the same
internal (primarily KCH3O3S) and external
(calcium Tyrode's) solutions as for the current-clamp recordings,
designed to approximate physiological ionic conditions. Voltage steps
positive to 55 mV activated inward current, which reached a maximum
for steps near 0 mV and was followed by outward current for steps
beyond 30 mV. TTX (300 nM) completely blocked the inward
current evoked by steps to all voltages, leaving large outward
currents. With the spike train waveform (Fig. 2B), under control conditions there was a large inward current that reached
a peak during the upstroke of each spike, followed by an outward
current that was largest during the falling phase of each spike. TTX
completely blocked the inward current elicited by the spike train.
Thus, the upstroke of the action potentials is caused entirely by
TTX-sensitive sodium current, with no contribution of voltage-dependent
calcium current.
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Figure 2.
Sodium currents during voltage steps and spikes in
Purkinje cell bodies. A, Holding potential, 60 mV.
Steps were 60 to 15 mV in 5 mV increments (top
panel). Raw current evoked by step depolarizations in
calcium Tyrode's (top) and in calcium Tyrode's with
300 nM TTX (middle). The difference between
the two sets of traces shows the TTX-sensitive sodium current
(bottom). B, Sodium current evoked by a
train of action potentials. Same cell as in A. The spike
train protocol was used as the voltage-clamp command waveform
(top panel). Raw and subtracted traces as in
A. The command waveform was presented five times with a
1 sec interval, and the resulting currents were averaged. In this and
all figures of currents evoked by the spike train protocol, the
dotted line indicates 0 pA. To facilitate comparison,
vertical gain is the same as that in A.
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In Figure 2, the peak sodium current during the action potential
upstroke (4.0 nA, reached at a voltage of 34 mV), was smaller than
the peak sodium current elicited by voltage steps (5.6 nA, reached at
a voltage of 15 mV). Because the steps were delivered from a holding
potential of 60 mV, more positive than the main part of the
interspike interval, it is unlikely that the smaller current during the
spike reflects more initial inactivation. Most likely, activation of
sodium channels is not complete during the upstroke of the action
potential. Even more striking is the difference in magnitude of the
outward current elicited by a spike compared with that activated by 12 msec steps. In the experiment shown in Figure 2, the peak outward
current during the spikes (which reach a peak of +14 mV) is 5.7 nA,
whereas outward current elicited by the steps exceeds 20 nA for a step
to 15 mV. Only a very small fraction of available potassium
conductance is activated during a spike.
In many cells, it was impossible to adequately voltage clamp the sodium
currents. When elicited from a holding potential of 90 mV, sodium
currents were almost always larger than 20 nA, the level at which the
headstage amplifier reached saturation, and they often exceeded 20 nA
even from a holding potential of 65 mV, where nearly half the
channels are inactivated (Raman and Bean, 1997). We assessed quality of
clamp by a smoothly graded increase in the sodium currents evoked in
the region of 50 to 20 mV by steps in 5 mV increments. By this
criterion, we obtained good clamp of sodium current in five neurons for
which we could obtain a complete series of recordings with and without
TTX. The neuron whose records are shown in Figure 2 is one of these. A second criterion for good voltage clamp was the behavior of the TTX-sensitive currents elicited by step depolarizations, obtained by
subtracting currents remaining in TTX from control records (Fig.
2A, bottom panel). In cells with
imperfect voltage control, the expected transient inward current was
followed by small transient outward currents. These outward currents
are expected from small errors in voltage control
for example, those
caused by incompletely compensated series resistance. In control
solution, the large inward sodium current produces an additional
depolarization by its flow through the series resistance, and this
larger depolarization activates a larger outward current than that
activated by the same nominal voltage step without inward sodium
current. Because the outward current during a 12 msec step
depolarization changes dramatically for a 5 mV increment in test
voltage (>5 nA between 20 and 15 mV for the cell in Fig. 2), a
change in outward current with TTX application is a very sensitive
assay for voltage-clamp errors. In the five neurons with optimal
voltage control, either there were no TTX-sensitive outward currents
(as in Fig. 2) or they were small enough to be consistent with voltage
errors of only 1-2 mV.
Subtracting the currents elicited by the spike train before and after
TTX application yields the sodium current flowing during the
spontaneous activity (Fig. 2B, bottom). In
the five neurons with optimal voltage control, this current was inward
throughout the spike train. In these five cells, the peak sodium
current during the upstroke of a spike was 6.0 ± 1.2 nA. This
can be compared with the ionic current flowing during the upstroke of spontaneous spikes, which can be calculated from the product of cell
capacitance and dV/dt, the time derivative of
voltage (Hodgkin and Huxley, 1952). The maximal rate of depolarization
was 255 ± 25 mV/msec, which would correspond to 6.4 ± 0.6 nA for a typical 25 pF cell. Thus, the measured sodium current can
account well for the upstroke velocity of spontaneous action potentials.
Figure 3 shows at higher resolution the
TTX-sensitive sodium current elicited by the spike train. There is
sodium current flowing at all times between spikes (Fig.
3A,B). The current between spikes had a characteristic
kinetic feature consisting of a secondary rise in current that occurred
when the action potential reached its trough (Fig. 3C). This
is reminiscent of the "resurgent" sodium current that occurs on
repolarization after strong depolarizations in both rat and mouse
Purkinje neurons (Raman and Bean, 1997; Raman et al., 1997; Kay et al.,
1998). The decay of this secondary rise in current is consistent with
the decay kinetics of resurgent current (time constant of ~3 msec at
70 mV) (Raman and Bean, 1997). After the decay of this component, the
interspike sodium current typically reached a minimum near 65 mV and
then increased in a regenerative manner. In the five cells with optimal
voltage clamp, the mean TTX-sensitive sodium current was 22 ± 3 pA between 70 and 65 mV, and 48 ± 4 pA between 65 and
60 mV. There was a smooth transition from the small sodium current
between spikes to the large current during the upstroke of a spike.
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Figure 3.
Kinetics of sodium currents evoked by spike train
protocol. A, Command voltage and TTX-sensitive sodium
currents (different cell than in Fig. 2). Currents were obtained by
subtraction as in Figure 2. B, Display at faster time
base and greater magnification of command voltage and current response
within dashed lines in A.
C, First 13 msec of the trace in B. The
arrow indicates the bump of sodium current that occurs
when the action potential command reaches its trough.
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Calcium currents and potassium currents
We next examined the possible contribution of voltage-dependent
calcium channels to spontaneous firing. Isolated Purkinje neurons have
large voltage-dependent calcium currents (Kaneda et al., 1990; Regan,
1991; Mintz et al., 1992; McDonough and Bean, 1998), which often
exceeded 1 nA with physiological external calcium (e.g., see Fig. 8).
Replacement of calcium by cobalt rapidly and reversibly blocks calcium
current through all types of calcium channels. To examine calcium
currents in physiological solutions, we recorded currents in calcium
Tyrode's solution + 300 nM TTX (Fig.
4C,D) and in cobalt Tyrode's
solution + 300 nM TTX (Fig. 4E,F).
The difference between the traces with calcium and with cobalt consists
of calcium current plus any currents activated by the calcium entry.
Calcium currents in Purkinje cells are large and sustained (Regan,
1991; Mintz et al., 1992). However, during the step depolarizations in
these solutions, the net cobalt-sensitive current had only a barely
detectable phase of inward current that is rapidly overwhelmed by
outward current (Fig. 4G). This outward current is likely to
be calcium-activated potassium current, because the tail currents after
step depolarizations reversed near EK and
changed as expected on increasing external potassium from 4 to 16 mM (data not shown). Remarkably, in response to the spike train command, the current blocked by cobalt substitution was net
outward at all times during spikes, and it decayed to zero between
spikes (Fig. 4H,I). Thus, at no time during
the pacemaking cycle does calcium entry appear to have a net
depolarizing effect.
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Figure 4.
Calcium-dependent and -independent currents during
steps and spikes in a Purkinje cell body. A, Step
voltage protocol for C, E, and G; holding
potential, 90 mV; steps, 60 to 0 mV, 10 mV increments.
B, Spike train protocol for D, F, and
H. C, D, Raw currents evoked in calcium
Tyrode's with 300 nM TTX. Calibration in C
also applies to E and G. Calibration in
D also applies to F and H,
and vertical gain is twice that in C, E, and
G, to facilitate comparison. E, F,
Currents evoked in cobalt Tyrode's with 300 nM TTX.
G, H, Cobalt-sensitive current, obtained by subtraction
of records in cobalt Tyrode's from those in calcium Tyrode's.
I, Amplification of current within dashed
lines in H, to illustrate the lack of
cobalt-sensitive current in the interspike interval.
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Low concentrations of external TEA are effective in blocking some
potassium currents in Purkinje neurons (Gähwiler and Llano, 1989;
Gruol et al., 1991). Accordingly, we assessed the outward currents'
sensitivity to 1 mM TEA. Figure
5 shows currents from the same cell
illustrated in Figure 4. In Tyrode's solution containing 1 mM TEA (as well as 300 nM TTX), step
depolarizations elicited an early inward phase of current that quickly
became outward, presumably consisting of calcium current followed by
TEA-resistant potassium current. With calcium Tyrode's solution, 1 mM TEA reduced the peak outward current elicited by a step
to 0 mV by 83 ± 2%, and the steady-state current measured at the
end of the 12 msec step by 63 ± 3% (n = 8) (Fig.
5C vs 4C). When recordings were repeated in
cobalt Tyrode's solution, the inward current disappeared, and the
outward currents were reduced in amplitude and activated at a much
slower rate (Fig. 5E).
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Figure 5.
TEA-sensitive currents during steps and spikes in
a Purkinje cell body. Same cell as in Figure 4. Calibrations for
responses to step protocol (A) and spike train
protocol (B) are the same as in Figure 4. Voltage
protocol in A applies to C, E, G, and
I. Steps indicated by the dashed lines
are to +10 and +20 mV and apply only to the dashed
traces in I. Voltage protocol in
B applies to D, F, H, and
J. C, D, TEA-insensitive currents. Raw
currents evoked in calcium Tyrode's with 1 mM TEA and 300 nM TTX. E, F, TEA-insensitive,
calcium-independent currents. Raw traces evoked in cobalt Tyrode's
with 1 mM TEA and 300 nM TTX. G,
H, Total TEA-sensitive current. Currents in C
and D subtracted from those in Figure 4,
C and D, respectively. I,
J, TEA-sensitive, calcium-independent current. Currents in
E and F subtracted from those in Figure
4, E and F, respectively. The
dashed traces in I show currents evoked
at more positive potentials and illustrate the rapidly inactivating
component of TEA-sensitive calcium-independent current.
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Although TEA blocked outward currents elicited by step depolarizations
only partially, it abolished all outward current elicited by the spike
train command (Fig. 5D), leaving only an inward current that
could be blocked by cobalt (Fig. 5F). This
observation was consistent across cells (n = 8). Thus,
despite the multiple types of potassium currents present in the cells,
those evoked during a spike are all TEA sensitive.
The TEA-sensitive currents measured in calcium therefore represent the
total potassium current evoked by the spike train command. The
potassium current activated by each spike was largest during the
falling phase of the spike, as expected, and it then decayed very
rapidly on repolarization (Fig. 5H,J). Fitting the
decay of the total TEA-sensitive current with a single exponential gave a time constant (
) of 0.28 ± 0.01 msec (n = 6), and the total potassium current between spikes was essentially zero
(1.1 ± 1.8 pA, n = 6, measured between 65 and
60 mV). The decay of potassium current must reflect rapid and
complete deactivation of channels and not simply a decrease in
driving force, because the troughs of the spike train are still 25 mV
positive to EK.
In response to step depolarizations, both calcium-dependent and
calcium-independent components of the TEA-sensitive current show rapid,
partial inactivation (Fig. 5G,I). The total
TEA-sensitive current inactivates by 35 ± 3% during the 12 msec
step (n = 8). Consistent with inactivation of
calcium-dependent potassium current, 100 nM iberiotoxin
blocked a current that inactivated partially with a time constant of 3 msec at 0 mV (n = 4; data not shown). The TEA-sensitive
current recorded in cobalt (Fig. 5I) shows rapid activation and a rapidly inactivating phase positive to 0 mV, with an
exponential decay constant of 2.3 ± 0.2 msec at +10 mV (n = 7), which decreased to 1.6 ± 0.3 msec at +30
mV (n = 4).
Figure 6 shows the voltage dependence of
various components of potassium current. Stepping from 90 mV, the
first measurable currents (
10 pA) were at or slightly positive to
50 mV for calcium-dependent potassium current (n = 8)
and were at 40 mV for the calcium-independent potassium current
(n = 6). Thus, there is insignificant activation of any
of the components of potassium currents at voltages in the main part of
the interspike interval (70 to 55 mV). This is consistent with the
lack of TEA-sensitive potassium current between spikes.
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Figure 6.
Current-voltage relations for outward currents
evoked by step depolarizations. All currents were recorded in 300 nM TTX. Data from the traces illustrated in Figures 4 and
5. Ca, Calcium Tyrode's; Co, cobalt
Tyrode's; CaTEA, calcium Tyrode's + 1 mM
TEA; CoTEA, cobalt Tyrode's + 1 mM TEA.
Current was measured as the maximal outward current during the 12 msec
step. A, Raw currents in the solutions indicated.
B, Difference currents for the solutions
indicated.
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To isolate overall calcium current evoked by the spike waveform, we
looked at the difference between recordings made in calcium with TEA
and cobalt with TEA, which is shown in Figure
7. This revealed a transient of calcium
current that reached a maximum during the falling phase of the action
potential. Although the step depolarizations showed inward current
followed by outward current, indicative of a substantial
calcium-dependent potassium current not blocked by 1 mM
TEA, no outward current was ever seen in response to the spike train
command. The lack of current between spikes under these conditions
indicates that not only is the net calcium current plus
calcium-dependent potassium current near zero between action potentials
(Fig. 4I), but each of these currents by
itself is also near zero (Fig. 7E).
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Figure 7.
Cobalt-sensitive, TEA-insensitive currents during
steps and spikes in a Purkinje cell body. Same cell as in Figures 4 and
5. Calibrations for response to step protocol (A)
and spike train protocol (B) are the same as in
Figures 4 and 5. C, Currents in Figure 5E
subtracted from those in Figure 5C. D,
Currents in Figure 5F subtracted from those in Figure
5D. Note that all current is inward in response to the
spike train protocol, despite the outward current in C.
E, Magnification of current within dashed
lines in D, to illustrate the lack of interspike
current.
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To identify the calcium channels producing the transient of calcium
current during the spike train, we examined the pharmacology of these
currents. To isolate calcium currents, these recordings were made with
a CsCl-based intracellular solution and external solution of calcium
Tyrode's solution + 300 nM TTX. -Conotoxin-GVIA (at 3 µM) blocked little or no current in mouse Purkinje
neurons (data not shown; n = 3), consistent with
previous estimates that N-type current contributes only ~5% of
overall high-threshold current in rat Purkinje neurons (Regan, 1991;
Mintz et al., 1992). Consistent with this, after application of 100 nM -agatoxin-IVA, there was no further block of current
by 1 µM -conotoxin-MVIIC, which blocks N-type as well
as P-type channels (McDonough et al., 1996). We were therefore able to
use -conotoxin-MVIIC rather than -agatoxin-IVA (of which we had
limited amounts) to identify P-type current. Recordings were made first
in calcium Tyrode's solution and then with -conotoxin-MVIIC. Other
calcium channel toxins were then added sequentially and cumulatively:
500 nM mibefradil was used to isolate low-threshold
(T-type) calcium current (McDonough and Bean, 1999), and 2 µM nimodipine was used to isolate L-type current.
Finally, recordings were repeated in cobalt Tyrode's solution to block
any remaining calcium current. This sequence avoided effects of
mibefradil on P-type current, which are small (McDonough and Bean,
1998), or nimodipine on T-type current, which can be significant
(our unpublished results). The individual calcium currents
isolated in this way are shown in Figure
8. The characteristics of P-type and
T-type currents elicited by step depolarizations (Fig.
8A) are as expected (Regan, 1991; Mintz et al., 1992;
McDonough and Bean, 1998): T-type currents have slower tail currents
and activate slightly more negatively (Fig. 8C). L-type
current and current remaining with -conotoxin-MVIIC, mibefradil, and
nimodipine were both very small in all cells tested.
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Figure 8.
Pharmacologically segregated calcium currents
during steps and spikes in Purkinje cell bodies. Currents were recorded
with intracellular CsCl. Extracellular solutions contained 300 nM TTX. Sequence of (cumulative) addition of blockers was 1 µM -conotoxin-MVIIC, 500 nM mibefradil,
and 2 µM nimodipine. Then recordings were repeated in
cobalt Tyrode's solution to block the small amount of calcium current
remaining. A, Step protocol (top
panel) and evoked calcium currents.
MVIIC-sensitive, mibefradil-sensitive,
nimodipine-sensitive, and cobalt
sensitive designate currents blocked by each agent, as obtained
by subtraction of records with and without the blocker.
B. Spike train protocol (top
panel), and currents evoked by the same combinations of
blockers as in A, for the same cell as in
A. Vertical gain on currents is twice that in
A. C, Current-voltage relation for
MVIIC-sensitive (P-type) and mibefradil-sensitive
(T-type) current, showing the more negative activation
of T-type current. D, Mibefradil-sensitive currents
evoked by the spike train protocol with different intersweep holding
potentials. In the top current trace, the cell was held
at 65 mV for 1 sec between applications of the spike train protocol.
In the bottom current trace, the cell was held at 95
mV for 1 sec between applications of the same protocol. Notice the
onset of the mibefradil-sensitive current before the large slow tail in
response to the first spike stimulus.
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The calcium currents evoked by the spike train command are shown in
Figure 8B. The dominant component was a large,
rapidly decaying P-type current (Fig. 8B). In some
cells, a much smaller T-type current was also present. In four cells,
measuring between 70 and 65 mV, T-type current was 7 ± 3 pA, decreasing to 4 ± 2 pA between 65 and 60 mV. The
small T-type current is caused mainly by steady inactivation during the
spike train, because if the cell was held at 95 mV before applying
the spike train (instead of the usual 65 mV), slowly decaying
currents between spikes were evident (Fig. 8D).
However, nearly complete inactivation set in by the sixth spike in the
train. Thus, T-type currents are unlikely to dominate the interspike
current, although their contribution will be sensitive to the extent of
hyperpolarization between spikes.
Ih
Spontaneous firing of many excitable cells is promoted by
Ih, a voltage-dependent current activated
by hyperpolarization (Mayer and Westbrook, 1983; McCormick and Pape,
1990; Pape, 1996). Ih is present in Purkinje
neurons studied in brain slices (Crépel and Penit-Soria, 1986).
We found that isolated Purkinje neurons also possessed
Ih. Hyperpolarizing voltage steps elicited
characteristic time-dependent currents that grew over a period of
hundreds of milliseconds and were blocked by 1 mM cesium in
the external solution (Fig.
9A). The maximal amplitude of
Ih current was 48 ± 5 pA at 120 mV
(n = 7). In response to the spike train protocol, there was no cesium-sensitive current during the interspike intervals (Fig.
9B), consistent with the requirement for more negative
voltages (beyond 80 mV) for significant activation of
Ih. The small cesium-sensitive outward currents
during the spikes most likely reflect weak block of
depolarization-activated potassium currents.
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Figure 9.
Cesium-sensitive currents in a Purkinje cell body.
This is the same cell as in Figures 4-7. A, Step
protocol and cesium-sensitive (hyperpolarization-activated) currents
obtained by subtraction of recordings in calcium Tyrode's with and
without 1 mM CsCl; intracellular
KCH3O3S solution; holding potential 70 mV;
steps from 90 to 120 mV, in 10 mV increments. B,
Spike train protocol and resulting currents for same solutions as in
A.
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Current-clamp experiments
We next examined effects of various channel blockers on
spontaneous firing. As illustrated in Figure
10A, cesium (1 mM) had no effect on spontaneous firing rates (102 ± 4% of control; n = 12), although cesium did block the
sag in the membrane voltage in response to hyperpolarizing current
pulses, which is characteristic of Ih (Fig.
10B). These recordings also illustrated the high
input resistance of the Purkinje cell bodies. In steps from 65 mV
with calcium Tyrode's solution, the input resistance was 900 ± 75 M (n = 7).
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Figure 10.
Spontaneous firing in the presence and absence of
cesium. A, Spontaneous firing in calcium Tyrode's
(left) and in calcium Tyrode's with 1 mM
cesium (right). The spiking frequency changed from 52 to
46 Hz. B, Voltage-current relation in the same cell as
A, in the same solutions. Step hyperpolarizations were
given from 0 pA to 0, 20, 60, and 100 pA (bottom
panels). In the presence of cesium, the
hyperpolarization-induced sag in membrane potential was abolished, and
there was a slower return to baseline after the offset of the
hyperpolarizing pulse.
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To investigate whether the small T-type current evoked under voltage
clamp exerts a measurable effect on firing, we applied 500 nM mibefradil to spontaneously firing neurons. In
mibefradil, the rate of firing decreased (Fig.
11A) and on average
was slowed to 72 ± 3% of the control frequency in calcium
Tyrode's solution (n = 5). These results suggest some
participation of T-type channels in shaping spontaneous firing.
However, we cannot be completely certain that mibefradil affected only
T-type calcium channels in the current-clamp experiments. Although
mibefradil is highly selective for T-type over P-type calcium channels
(McDonough and Bean, 1998), it has not been systematically tested
against other kinds of voltage-dependent channels.
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Figure 11.
Effects of blocking calcium currents on action
potential firing. Traces in A-D are from
different Purkinje neurons. A, Spontaneous action
potentials recorded in calcium Tyrode's and calcium Tyrode's with 500 nM mibefradil, as marked. The firing rate was reduced from
25.5 Hz in control to 19.5 Hz in mibefradil. B,
Spontaneous activity in calcium Tyrode's and cobalt Tyrode's. The
frequency of spiking changed from 52 to 35 Hz. Same cell as in Figure
10. C, Responses of a spontaneously active cell that was
silenced in cobalt Tyrode's. The cell rested at 45 mV
(left). With a constant injection of 10 pA
hyperpolarizing current, the cell fired repetitively and regularly
(middle). With injection of 20 pA hyperpolarizing
current, the cell was silenced again, resting at 85 mV
(right). D, Responses of another
spontaneously active cell that was silenced in cobalt Tyrode's. After
a 100 msec, 50 pA hyperpolarization, the cell fired a short train of
action potentials, which showed a progressive reduction in the depth of
the hyperpolarization and the peak of the depolarization.
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When all calcium currents (and calcium-activated currents) were blocked
by cobalt Tyrode's solution, 17 of 21 cells continued to fire
spontaneously (Fig. 11B). On average, cobalt reduced
the firing rate to 83% of control. However, the change in rate was variable and sometimes the firing rate actually increased (range, 34 to
158% of control); an increase in stimulus-evoked firing rate with
nickel block of calcium channels has been reported previously in intact
Purkinje neurons (Callewaert et al., 1996). Four of the 21 cells
studied ceased to fire in cobalt. On cessation of firing, these cells
rested at a relatively depolarized membrane potential of 46 ± 1 mV. This is consistent with the voltage-clamp results showing that the
dominant electrical effect of calcium influx is to activate an outward
current, which would promote hyperpolarization. Thus, silencing is not
attributable to removal of depolarizing calcium current but rather to
depolarization block. In fact, with continuous application of 
10 pA
of hyperpolarizing current, three of these cells resumed spontaneous
firing, as shown in Figure 11C. The fourth cell could be
induced to fire a short series of action potentials after brief
hyperpolarizations, as shown in Figure 11D. Silencing
by cobalt had a characteristic profile of spike trough becoming less
negative and spike peak potential becoming less positive for several
spikes preceding the cessation of firing. This suggests that, in these
cells, calcium-dependent potassium current is important for
hyperpolarizing the membrane potential sufficiently for substantial
recovery of sodium channels. The majority of cells, which maintain
spontaneous activity in cobalt, may have enough calcium-independent
potassium current to repolarize the cell for sufficient recovery of
sodium channels between spikes, or large enough sodium currents so that
firing can persist even with a reduced availability of sodium channels.
As illustrated in Figure
12A, 300 nM TTX blocked firing in all cells tested
(n = 10). The resting potential in 300 nM
TTX was 62 ± 3 mV (n = 10), and no underlying
fluctuations or oscillations in membrane potential remained. In 300 nM TTX, depolarizing current did not elicit action
potentials, nor did hyperpolarizations produce rebound
low-threshold calcium spikes (Fig. 12B).
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Figure 12.
Effects of TTX on spontaneous firing and membrane
potential. A, Same cell as in Figure 10 and
11A. Firing was abolished in 300 nM
TTX, and the cell rested at 64 mV. B, Voltage-current
relation in 300 nM TTX; steps from 100 to +100 pA in 20 mV increments. C, Spontaneous firing in calcium
Tyrode's, with 0, 3, and 10 nM TTX, as indicated. This
cell continued to fire for some time in 10 nM TTX
(early) before silencing and resting at 51 mV
(late).
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We also examined the effect of submaximal blocking concentrations of
TTX on firing. A dose-response relation for TTX block of peak
transient sodium currents under voltage clamp identified the
half-maximal blocking concentration to be 2.5 nM,
consistent with the estimate of 3 nM for block of sodium
current in guinea pig Purkinje neurons (Kay et al., 1998). Although we
did not directly examine the potency of TTX on interspike sodium
currents, this is likely to be the same as for peak transient current,
because Kay and colleagues (1998) found identical TTX sensitivity for inactivating and noninactivating sodium currents. All cells still fired
spontaneously in 3 nM TTX (~60% block of available
channels), as illustrated in Figure 12C (n = 7). The firing rate fell to 70 ± 6% of the control rate, and
peak membrane potential of the spontaneous spikes decreased by 12 ± 3 mV.
In 10 nM TTX (~90% block), six of seven cells tested
were silenced. Figure 12C shows the last two sweeps of
firing and silencing in a cell exposed to 10 nM TTX. The
spike peak and trough potentials were sequentially diminished before
the cessation of firing. As in the block sometimes induced by cobalt,
the silenced cells rested at a relatively depolarized membrane
potential of 53 ± 2 mV (n = 6), significantly
more positive than the resting potential 62 ± 3 mV in 300 nM TTX (p = 0.015; unpaired
t test). This suggests that even with 90% blockade of
sodium channels, the remaining sodium current can depolarize the
membrane potential by ~10 mV.
We next examined whether spontaneous firing could be stopped by an
interruption in the cycle of depolarization and repolarization. In
calcium Tyrode's solution, we voltage-clamped cells at various potentials for 4-6 sec to allow currents to equilibrate and then released the voltage clamp. After conditioning (voltage-clamped) periods at potentials ranging from 85 to 10 mV (in 5 mV
increments), all cells resumed firing within a few milliseconds of
release of the voltage clamp (n = 7; spontaneous firing
rates ranging from 17 to 121 Hz). As illustrated in Figure
13, the direction and magnitude
of membrane polarization on release of the voltage clamp depended on
the conditioning potential. Firing resumed regardless of the initial
direction of polarization; however, after the most positive
conditioning periods, the rate of firing was sometimes slightly reduced
at first. Occasionally this change was accompanied by a decrease in the
depth of the trough hyperpolarizations. The potential at which the
initial direction of membrane polarization reversed on release of
voltage clamp was 48 ± 1 mV (n = 7).
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Figure 13.
Absence of depolarization block; responses of a
Purkinje neuron that was voltage-clamped at (nominally) 80, 70,
60, 50, 40, 30, 20, and 10 mV (as labeled) for 4 sec. The
clamp was then released at the time point indicated by the
arrow, by switching into bridge mode (see Materials and
Methods). In all cases the cell resumed firing.
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This value is interesting for two related reasons. First, judging from
the voltage-clamp results, the overall sum of the different ionic
conductances at steady state is probably near a minimum between 40
and 50 mV. In this range of potentials, sodium and T-type calcium
channels are substantially inactivated, and potassium channels and
high-threshold calcium channels are at the foot of their activation
curves. Second, under current clamp, the cells that were silenced by
moderate blockade of sodium channels (and those few silenced by full
blockade of calcium channels) tended to rest in this voltage range.
Thus, we investigated whether cells could be silenced by forcing them
to particular voltages in this range of potentials. In general, this
was not possible. However, near the voltage at which membrane
polarization reversed, it sometimes took several hundred milliseconds
for firing to resume. In these cases, release of the clamp was followed
by an oscillation in membrane voltage that increased in amplitude until
firing resumed, as illustrated in Figure
14. When the potential was found with the slowest resumption of firing, the initial direction of voltage change was often variable. In Figure 14B, the cell
was clamped at 49 mV; in the trial shown in the top trace it
hyperpolarized on release of the clamp, and in that shown below, it
depolarized. The variability may reflect stochastic behavior of the few
channels active at this voltage. On rare occasions, cells ceased firing after clamping at or near the "reversal"; however, such silencing tended to occur late in the recordings and was difficult to distinguish from deterioration of the quality of the recording.
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Figure 14.
Oscillations of membrane potential in the region
of minimal currents. A, The cell was clamped at 45 mV
(top panel) or at 43 mV (bottom
panel). The initial direction of polarization on the
release of clamp was different and at each of these potentials was
consistent across trials (10/10, each condition). Arrow
indicates release of clamp. Calibration applies to both
A and B. B, Same cell as
in Figure 13. In both
panels, the cell was held at 49 mV for 6 sec before the clamp
was released. Notice the difference in the direction of the initial
polarization on release of clamp as well as the difference in the
duration of the oscillation.
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DISCUSSION |
These results show that the high-frequency spontaneous firing of
Purkinje neuron cell bodies depends mainly on TTX-sensitive current
flowing at voltages of 70 to 55 mV, as well as a high input
resistance and essentially no activation of voltage-dependent potassium
currents at these voltages.
In contrast to pacemaking in many other central neurons and in heart,
the hyperpolarization-activated cation current
Ih is not important for spontaneous firing of
Purkinje cells, at least in isolated cell bodies. Although the cells
possessed Ih, voltages during spontaneous
firing are never negative enough for significant activation.
Ih may be more important in intact neurons,
because the channels may be at a higher density in dendrites (Magee,
1998), consistent with much larger Ih currents
in intact cells [Crépel and Penit-Soria (1986); also see Budde
et al. (1994)]. Ih activation might be promoted
by transient hyperpolarization, for example from inhibitory synaptic
input, which is ongoing in intact cells (Häusser and Clark,
1997). Nevertheless, the cells can clearly fire at up to 150 Hz without
activation of Ih.
Calcium current and calcium-activated potassium current
T-type calcium current may play a secondary role in spontaneous
firing, because mibefradil slowed firing by ~30% but never stopped
it. Like Ih, T-type calcium current might
play a larger role in pacemaking if more hyperpolarized voltages were
reached as a result of inhibitory synaptic input.
Purkinje neurons have large P-type calcium currents. However, with
physiological ionic conditions, calcium entry during action potentials
is so tightly coupled to activation of calcium-activated potassium
current that the net current attributable to calcium entry is outward
at all times. Consistent with this, we could not elicit somatic calcium
spikes in normal Tyrode's solution when sodium current was blocked
with 300 nM TTX; however, reduction of calcium-activated
potassium current by 1 mM TEA enabled calcium spikes to be
elicited (n = 6; data not shown).
The tight coupling between calcium and calcium-dependent potassium
currents may contribute to the Purkinje cells' resistance to
depolarization block. The slow inactivation of P-type calcium channels
(Regan, 1991) would maximize calcium-dependent potassium currents with
steady depolarizations, thus promoting repolarization. Indeed, when
firing sometimes stopped in cobalt Tyrode's solution, it appeared to
be caused by block of the calcium-dependent potassium currents,
resulting in depolarization block.
Total cobalt-sensitive current was outward or zero at all times during
the spike train. However, we did not examine separately the ability of
current through T-type and P-type channels to activate potassium
current. The slowing of firing by mibefradil is consistent with a net
depolarizing effect of T-type current during the interspike interval.
This could occur if calcium current through T-type channels were not
effective at activating potassium currents.
Potassium currents
The calcium-dependent and -independent potassium currents that
flow during the spike train were both sensitive to 1 mM
TEA. Interestingly, in cultured Purkinje cells a developmental increase in the TEA sensitivity of potassium currents coincides with the onset
of regular spontaneous firing (Gruol and Franklin, 1987; Yool et al.,
1988). Step depolarizations elicited an additional component of
TEA-insensitive potassium current (cf. Wang et al., 1991), which
apparently has slower activation kinetics. Of various potassium
conductances described by single channel recording from Purkinje
neurons, some show inactivation (Yool et al., 1988; Gähwiler and
Llano, 1989; Gruol et al., 1991), consistent with the rapidly inactivating component of both calcium-dependent and -independent macroscopic potassium currents (Figs. 4, 5).
Sodium current properties
The interspike depolarization appears to depend mainly on sodium
current. Its properties, including TTX sensitivity, are consistent with
sodium current at subthreshold voltages previously characterized by
long voltage steps and slow voltage ramps (Kay et al., 1998). Both
spike-evoked and long-lasting sodium currents seem to be mainly in the
cell body of Purkinje neurons (Callaway and Ross, 1997), as originally
postulated by Llinás and Sugimori (1980a,b).
In response to the spike train protocol, we measured an average of
22 ± 3 pA of sodium current between 70 and 65. This sodium
current represents <0.1% of the maximal current that can be elicited,
but it is still enough to depolarize the cell fairly quickly. During
spontaneous firing, the rate of depolarization between 70 mV and 65
mV was 1.2 ± 0.3 mV/msec (n = 12), which would
correspond to a net ionic current of 30 ± 8 pA for a cell with
a typical capacitance of 25 pF. Between 65 and 60 mV, the rate of
depolarization was 1.7 ± 0.3 mV/msec, corresponding to net ionic
current of 42 ± 8 pA. This matches reasonably well with the
measured sodium current of 48 ± 4 pA between 65 and 60 mV.
Spontaneous firing that depends on subthreshold TTX-sensitive sodium
but not Ih or calcium current has been described
previously in suprachiasmatic nuclei neurons (Pennartz et al., 1997)
and in dopaminergic amacrine cells (Feigenspan et al., 1998). Purkinje cells fire at rates that are 10-50 times higher than either of these.
To maintain such high rates, distinctive kinetic properties of sodium
channels in Purkinje neurons may be important. The surge of sodium
current immediately after a spike, which may correspond to resurgent
current seen with step depolarizations, would speed the initial phase
of depolarization. Both the resurgent current and most of the steady
current at 70 to 50 mV are probably from the Scn8a
sodium channel, and mice lacking this channel show inconsistent pacemaking (Raman et al., 1997). Perhaps more important than the depolarizing effect of the resurgent current is its association with
rapid, partial recovery from inactivation at relatively depolarized voltages (12% recovery in 10 msec at 40 mV) (Raman et al., 1997). This quickly restores the availability of sodium channels for spike production.
Interaction of ionic currents to produce spontaneous firing
A key factor allowing spontaneous firing is the lack of active
potassium currents between 70 and 50 mV. Background potassium current is also small, because the input resistance is high. Thus, initially small sodium currents can depolarize the cell to threshold. The steep voltage dependence and high density of sodium channels enable
the depolarization to be strongly regenerative and relatively fast. The
potassium currents that repolarize the action potential in Purkinje
neurons are notable for their very fast deactivation, so that the cells
do not hyperpolarize very deeply. Unlike many other neurons, there is
not an afterhyperpolarization that approaches EK. The rapid deactivation of potassium current
also returns the input resistance to a high value within milliseconds,
so that the small interspike sodium current can effectively depolarize the cell for another action potential.
Some repetitive phenomena, like the swinging of a pendulum, can be
stopped by temporarily arresting the cycle at a particular position. In
principle, spontaneous firing of an excitable cell might require a
continuous cycle of interacting voltage-dependent conductances.
For example, generation of a full-blown spike might be needed to
activate potassium currents sufficient to produce a hyperpolarization
deep enough to allow recovery from inactivation of sodium channels. In
such a case, any interruption of the cycle might silence the cell. This
is clearly not the case in Purkinje neurons, because regular firing
always resumed after voltage clamping the cell at any voltage. It is
particularly notable that cells do not enter depolarization block.
Firing resumed even after voltage clamping the cell at 10 mV for 4 sec, which maximally inactivates sodium channels and partly inactivates
potassium channels. After the release of clamp, the remaining potassium
current hyperpolarized the membrane such that sodium channels recovered
enough to resume spontaneous firing. Thus, the basal state of
spontaneous endogenous firing can be quickly restored after large
perturbations (e.g., from synaptic inputs) in either direction.
The robustness of firing is also evident in the remarkably small
effects of various ionic substitutions and pharmacological manipulations. Even total block of the large calcium currents (and
secondary block of even larger calcium-activated potassium currents)
usually had little effect on firing rate. Equally surprising, partial
block of sodium channels by 3 nM TTX, which blocked ~60% of sodium channels, reduced the spontaneous firing frequency by only
~30%. Evidently, firing rate has a sublinear dependence on sodium conductance.
Comparison with intact neurons
The regular spontaneous firing of isolated Purkinje cell bodies is
strikingly similar to that of intact Purkinje neurons studied in brain
slices when both excitatory and inhibitory synaptic inputs are blocked
(Häusser and Clark, 1997). The similarity suggests a primary
importance of somatic currents for spontaneous firing, although the
firing of intact cells must also be influenced by the voltage-dependent
channels present in dendrites (Llinás and Sugimori, 1980a,b;
Usowicz et al., 1992; Stuart and Häusser, 1994), as well as by
the resistive and capacitative load of the dendritic tree (Jaeger et
al., 1997). It has long been appreciated from both experiments
(Llinás and Sugimori, 1980a,b; Hounsgaard and Midtgaard,
1988) and modeling (De Schutter and Bower, 1994a,b; Jaeger et
al., 1997) that the intrinsic membrane properties of Purkinje neurons
are likely to powerfully influence responses to synaptic inputs. The
high-frequency spontaneous firing of Purkinje neurons in the absence of
synaptic currents, or even dendritic currents, strongly reinforces this idea.
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FOOTNOTES |
Received Sept. 22, 1998; revised Dec. 17, 1998; accepted Dec. 21, 1998.
This work was supported by National Institutes of Health (NS36855).
I.M.R. was supported by National Research Service Award NS10396.
Correspondence should be addressed to Indira M. Raman, Department of
Neurobiology and Physiology, Northwestern University, 2153 North Campus
Drive, Evanston, IL 60208.
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Alger BE,
Nicoll RA
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Spontaneous inhibitory postsynaptic potentials in hippocampus: mechanism for tonic inhibition.
Br
Top
Abstract
Introduction
