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Volume 17, Number 12,
Issue of June 15, 1997
pp. 4517-4526
Copyright ©1997 Society for Neuroscience
Resurgent Sodium Current and Action Potential Formation in
Dissociated Cerebellar Purkinje Neurons
Indira M. Raman and
Bruce
P. Bean
Vollum Institute, Oregon Health Sciences University, Portland,
Oregon 97201, and Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02115
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Voltage-dependent sodium channels were studied in dissociated
cerebellar Purkinje neurons from rats. In whole-cell recordings, a
tetrodotoxin (TTX)-sensitive inward
current was elicited when the membrane was repolarized to voltages
between 
60 and 20 mV after depolarizations to +30 mV long enough to
produce maximal inactivation. At 40 mV, this "resurgent" current
peaked in 8 msec and decayed with a time constant of 30 msec. With 50 mM sodium as a charge carrier, the resurgent current was on
average ~120 pA. CA3 pyramidal neurons had no such current. The
current may reflect recovery of inactivated channels through open
states, because in Purkinje neurons (but not CA3 neurons) there was
partial recovery from inactivation at 40 mV, coinciding with the rise of resurgent current. In single-channel recordings, individual channels
gave openings corresponding to resurgent and conventional transient
current. Action potentials were recorded from dissociated neurons under
current clamp to investigate the role of the resurgent current in
action potential formation. Purkinje neurons fired spontaneously at
~30 Hz. Hyperpolarization to 85 mV prevented spontaneous firing,
and brief depolarization then induced all-or-none firing of
conglomerate action potentials comprising three to four spikes. When
conglomerate action potentials were used as command voltages in
voltage-clamp experiments, TTX-sensitive sodium current was elicited
between spikes. The falling phase of an action potential is similar to
voltage patterns that activate resurgent sodium current, and thus,
resurgent sodium current likely contributes to the formation of
conglomerate action potentials in Purkinje neurons.
Key words:
sodium channel;
Purkinje neuron;
complex spike;
afterdepolarization;
tetrodotoxin;
pacemaking;
single channel;
action
potential;
cerebellum
INTRODUCTION
Voltage-dependent sodium channels produce the
large, inward currents that underlie fast action potentials in neurons.
In addition, smaller currents flowing through the channels can
influence subthreshold electrical behavior, as suggested originally by
effects of tetrodotoxin (TTX) at subthreshold voltages (Hotson et al.,
1979
; Llinás and Sugimori, 1980a). Subsequent voltage-clamp
experiments in many types of central neurons have shown the existence
of persistent or noninactivating sodium currents active at subthreshold
voltages (Stafstrom et al., 1985; French et al., 1990; Cepeda et al.,
1995). Such currents may play major roles in regulating repetitive
firing, amplifying dendritic depolarization, and producing
afterdepolarizations and plateau potentials (for review, see Taylor,
1993; Crill, 1996). There is still little known about the channels that
underlie these currents. In particular, the question of how they are
related to the sodium channels that produce the large, inactivating
currents of the action potential has proven difficult (Crill,
1996).
Cerebellar Purkinje neurons have long been a focus of detailed
electrophysiological study. Two striking features of Purkinje neurons
in vivo are regular, spontaneous firing (Bell and Grimm, 1969; Latham and Paul, 1971) and, upon stimulation of climbing fibers,
formation of "complex spikes" consisting of multiple peaks (Eccles
et al., 1966,1967; Martinez et al., 1971). Work with in vitro preparations has suggested that both properties may depend, in part, on intrinsic membrane properties of Purkinje neurons. Spontaneous firing is maintained in cerebellar brain slice preparations (Hounsgaard, 1979; Llinás and Sugimori, 1980a) and in cultured Purkinje neurons (Gruol and Franklin, 1987), even when synaptic activity is blocked. Although complex spikes apparently occur only
after excitation of climbing fibers when Purkinje neurons are studied
in situ (Eccles et al., 1966; Stuart and Häusser, 1994), multipeaked action potentials are also seen in cultured Purkinje
cells in the absence of climbing fiber or other synaptic input (Gruol
and Franklin, 1987; Gruol et al., 1992).
Llinás and Sugimori (1980a,b) attributed the distinctive firing
properties of Purkinje neurons to a variety of intrinsic membrane
conductances in the neurons, including voltage-activated calcium
conductances and a persistent sodium conductance. On the basis of their
suggestion that a noninactivating sodium conductance is located in the
cell body of Purkinje neurons, we have used a preparation of
dissociated Purkinje neurons to study subthreshold sodium currents with
the goal of understanding the physiological importance of such
currents. We found unusual voltage- and time-dependent behavior of
sodium current in which repolarization from a voltage of +30 mV to
voltages near 40 mV elicits a transient current of several hundred
picoamps. This "resurgent" sodium current, which is sensitive to
TTX, is present in Purkinje neurons, but not present in CA3 pyramidal
neurons. Single-channel experiments suggest that the channels
underlying the resurgent sodium current also generate conventional
fast-inactivating sodium current. The resurgent sodium current likely
contributes to the tendency of Purkinje neurons to fire spontaneously
and form multipeaked action potentials, features that were preserved in
the isolated cells.
MATERIALS AND METHODS
Preparation of cells. Experiments were
performed on Purkinje and CA3 neurons isolated enzymatically from rat
cerebellum and hippocampus, respectively, with dissociation techniques
established in the laboratory (Mintz et al., 1992). All reagents were
obtained from Sigma (St. Louis, MO). Long-Evans rats (postnatal day
8-14 for Purkinje cells, and 6-10 for CA3 cells) were anesthetized with ether before decapitation. Tissue was dissected 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. For Purkinje cells, the vermal layer of
the cerebellum was removed and minced; for CA3 cells, the hippocampus was removed, and 400 µm slices were cut on a tissue chopper. In both
cases, the tissue was then transferred to 10 ml of dissociation solution containing 3 mg/ml protease XXIII, pH 7.4 with NaOH, at
37°C, with oxygen blown over the surface of the fluid, in which it
was incubated for 7 (Purkinje cells) or 9 (CA3 cells) min. After
incubation, the tissue was washed in warmed oxygenated dissociation solution containing 1 mg/ml bovine serum albumin and 1 mg/ml trypsin inhibitor and then was 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; the CA3
region was dissected from slices before trituration. Purkinje cells
were identified by their large diameter and characteristic pear shape
because of the stump of the apical dendrite. CA3 pyramidal cells were
identified on the basis of their pyramidal morphology.
Recording techniques. All recordings were made at room
temperature.
Whole-cell recordings. Borosilicate pipettes (1-5
M
) were filled with an internal solution containing (in
mM) 117 CsCl, 9 EGTA, 9 HEPES, 1.8 MgCl2, 14 Tris-creatine PO4, 4 MgATP, and 0.3 Tris-GTP, buffered to
pH 7.4 with CsOH, and recordings were made with an Axopatch 200A (Axon
Instruments, Foster City, CA). Series resistance was compensated at
>90%. A control external solution containing (in mM) 50 NaCl, 110 TEA-Cl, 2 BaCl2, 0.3 CdCl2, 10 HEPES,
buffered to pH 7.4 with NaOH, was applied through flow pipes to the
voltage-clamped cell. Although these solutions blocked most
K+ and Ca2+ currents, protocols were repeated
in external solution plus 300 nM TTX, and these recordings
were subtracted from the control records to isolate TTX-sensitive
Na+ current.
Cell-attached, single-channel recording. Recordings were
made with quartz pipettes (10-20 M) filled with (in mM)
140 NaCl, 20 TEA-Cl, 2 BaCl2, 0.3 CdCl2, 10 HEPES, buffered to pH 7.4 with NaOH. TEA-Cl and CdCl2 were
included to minimize current from openings of K+ and
Ca2+ channels. Cells were bathed in a solution containing
(in mM) 160 KCH3O3S, 2 MgCl2, 0.1 EGTA, 10 HEPES, buffered to pH 7.4 with KOH to
bring the resting potential near 0 mV. Assessment of one-channel patches was made on the basis of examination of transient channel openings. If no openings to multiples of the unitary amplitude occurred
in 200 sequential trials, the patch was considered to be a
single-channel patch. The high peak probability of opening (range,
0.3-0.6) facilitated identification of multichannel patches.
Current clamping. Borosilicate pipettes (3-5 M) were
filled with (in mM) 122 KCH3O3S, 9 EGTA, 9 HEPES, 1.8 MgCl2, 14 Tris-creatine PO4,
4 MgATP, and 0.3 Tris-GTP, buffered to pH 7.4 with KOH. Cells were
bathed in Tyrode's solution. Recordings were made with an Axoclamp 2A
(Axon Instruments). Passive and active changes in membrane voltage,
including action potentials, were evoked by injected current. The
recorded responses were then used to generate waveforms for whole-cell,
voltage-clamp experiments, which were conducted in the solutions
described above in whole-cell recordings.
Acquisition and analysis. Data were acquired and analyzed
with pCLAMP software (Axon Instruments) and plotted with Origin (Microcal, Northampton, MA). Data are presented as mean ± SD.
RESULTS
We began studying sodium currents in dissociated Purkinje
neurons by characterizing the features of conventional sodium currents elicited by single depolarizations. Figure 1 shows the
basic properties of sodium channel currents in Purkinje neurons and
compares them with currents in CA3 pyramidal neurons. The currents in
the two cell types are similar in voltage dependence, with a current
first detectable at voltages near 45 mV and maximal near 15 mV. In both cell types, the peak conductance could be fit well by a single Boltzmann function (Fig. 1C). The voltages for half-maximal
activation (Purkinje, 33 ± 8 mV; CA3, 27 ± 6 mV), and
the slope factors (Purkinje, 5.8 ± 1.1 mV; CA3, 5.9 ± 1.9 mV) were not different substantially in the two cell types. The voltage
dependence of inactivation was also similar in Purkinje and CA3
neurons. Determined with 100-200 msec prepulses, the midpoint of the
inactivation curve was 56 ± 6 mV with slope factor 6.9 ± 0.6 mV (n = 9) for Purkinje neurons, and 55 ± 3 mV with slope factor 6.4 ± 1.0 mV (n = 4) for CA3
neurons.
Fig. 1.
Transient sodium currents in Purkinje and
CA3 neurons. A, Currents evoked from a holding potential
of 90 mV by 50 msec steps to potentials between 80 and +50 mV in 10 mV increments are shown for a Purkinje cell (left) and a
CA3 cell (right). The break in the trace
represents 39 msec. B, Current-voltage relation for the
peak currents in A. The points are
connected by a spline function for clarity. C, Peak
conductance (filled circles) and steady-state inactivation curves (open circles) for the cells shown
in A. Conductance curves were fitted
(lines) with the Boltzmann function,
G = Gmax/(1 + exp[(V
Vh)/k]), where G is
conductance in nanosiemens, Gmax is the
maximal conductance, V is the step potential in mV,
Vh is the voltage for half-maximal
activation in mV, and k is the slope factor in mV. The
fitted Gmax, Vh,
and k for the Purkinje neuron were 76.4 nS, 32.3 mV,
and 5.5 mV, and for the CA3 neuron 66.7 nS, 28.4 mV, and 4.8 mV. The
inactivation curves were fitted by the Boltzmann function 1/(1 + exp(V
Vh)/k).
Vh and k values were 56.5
and 5.8 mV for the Purkinje cell (left), and 67 and 6.2 mV for the CA3 cell (right).
[View Larger Version of this Image (24K GIF file)]
Despite the similarity in the voltage dependence of activation and
inactivation, there were small differences in sodium current kinetics
between the two cell types. Sodium currents in Purkinje cells activated
and inactivated slightly faster than currents in CA3 neurons. For
example, the time constant of decay at +20 mV was 0.30 ± 0.05 msec (n = 9) in Purkinje neurons and 0.48 ± 0.06 msec (n = 7) in CA3 neurons. The kinetics of the sodium
currents in dissociated Purkinje neurons were in good agreement with
those recorded in Purkinje neurons cultured organotypically by
Gähwiler and Llano (1989).
We were particularly interested in characterizing noninactivating
components of sodium current. In both cell types, the current defined
by TTX-subtraction had a very small, but detectable steady-state component in most cells. The magnitude of the steady-state current relative to peak current was variable among different cells. For a step
to 30 mV, average steady-state current was 1.9 ± 0.7% of peak
current in Purkinje neurons (n = 12) and 4.4 ± 6.9% in CA3 neurons (n = 8). Although the magnitude of
steady-state current is highly variable in both neuronal types, if
anything, the current is smaller in Purkinje neurons.
To describe the voltage dependence of steady-state current, we used
voltage ramps. Slowly rising ramps promote inactivation of transient
currents and provide a useful measurement of the voltage dependence of
persistent sodium current. Because the sweep can be obtained within
seconds and because TTX is applied immediately afterward, it is
possible to define accurately the voltage dependence of the
steady-state current. Figure 2 shows TTX-sensitive
current elicited by a voltage ramp from 90 to +30 mV at 0.1 mV/msec. As in previous studies of persistent sodium currents (Stafstrom et al.,
1985; French et al., 1990; Brown et al., 1994; Cepeda et al., 1995),
the peak current was reached between 40 and 30 mV in both CA3
neurons and Purkinje neurons. To check whether the current was at
steady-state during the ramp, we stepped back to 30 mV after its
completion, reasoning that a smaller current after the ramp would
suggest that slow inactivation had continued to occur after the maximal
steady-state current was reached. Remarkably, in Purkinje neurons the
return to 30 mV elicited an inward current consistently much
larger than the current at 30 mV during the ramp (Fig.
2B). The current elicited on repolarization to 30 mV decayed exponentially (
= 34 msec) back to a level comparable to
the current at 30 mV reached during the ramp. We refer to this
current as "resurgent" sodium current, because it rises again after
a voltage protocol expected to produce maximal inactivation. Such a
current was seen in seven of seven Purkinje cells studied with this
protocol. In contrast, none of eight CA3 cells studied with the same
protocol had such a transient current on repolarization to 30 mV, as
shown in Figure 2C.
Fig. 2.
Sodium currents elicited by slow ramps in
Purkinje and CA3 neurons. The membrane voltage was ramped from 90 mV
to +30 mV at 0.1 mV/msec (A). Currents evoked by this
protocol were recorded in a Purkinje neuron (B) and a
CA3 neuron (C). In both cases, sodium current was
determined by subtracting control current from that in 300 nM TTX (which seemed to leave only leak and capacity current). In both the Purkinje neuron and CA3 neuron, the maximal current during the ramp occurred at 40 mV. The peak transient current
at 30 mV in the CA3 cell was 66% of that in the Purkinje cell, hence
the scaling of the ordinates.
[View Larger Version of this Image (25K GIF file)]
To investigate the basis for this unusual current, we stepped the
membrane potential in Purkinje cells directly to +30 mV to produce
maximal inactivation of transient sodium current and then repolarized
the membrane to various potentials. Repolarization to voltages between
20 and 60 mV elicited a large transient current in Purkinje neurons
(Fig. 3B) but not in CA3 neurons (Fig. 3C). With increasing repolarization, the resurgent current
was first detectable near 10 mV, was maximal near 30 to 40 mV, and became too small and too fast to be easily resolved negative to
80 mV. The resurgent current had a distinct rising phase followed by
a decay phase that could be fit well by a single exponential. At 30
mV, the time-to-peak was 8.0 ± 2.4 msec, and the time constant of
decay was 30 ± 7 msec (n = 12). Both the
time-to-peak and decay time constant were strongly voltage-dependent in
the range of 60 to 40 mV (Fig. 4).
Fig. 3.
Resurgent sodium current. A, Sodium
current was evoked by a 20 msec step from 90 to +30 mV, after which
the membrane was repolarized to voltages between 20 and 60 mV.
B, TTX-sensitive sodium current elicited by this
protocol in a Purkinje neuron. The transient current at +30 mV is
offscale (peak of 2 nA). Leak and capacity currents in 300 nM TTX were subtracted. C, TTX-sensitive sodium current elicited by the same protocol in a CA3 neuron. Peak
current at +30 mV was 1.3 nA (offscale). Scale bars apply to both sets
of traces.
[View Larger Version of this Image (16K GIF file)]
Fig. 4.
Voltage dependence of resurgent sodium
current. A, Peak resurgent sodium current (after 20 msec
step to +30 mV) versus repolarization potential for the Purkinje cell
in Figure 3. At 10 mV, there was no peak current, but the
steady-state current of 50 pA is plotted. B, Rise time
(time to peak) and decay time constants for the resurgent sodium
currents.
[View Larger Version of this Image (13K GIF file)]
The peak amplitude of the resurgent current at 30 mV was 124 ± 52 pA (n = 12), measured with 50 mM
sodium. Relative to the peak transient current elicited by a step from
90 to 30 mV, the peak resurgent current was 3.5 ± 2.0%
(n = 9). Although the peak resurgent current is small
compared with the peak transient current, the resurgent current flows
for a longer time because of its much slower decay kinetics (decay ~30 msec for resurgent current vs ~1.5 msec for transient current
at 30 mV). At 30 mV, the total charge transfer in the first 40 msec
of the repolarization-gated current was 31 ± 14% of the charge
transfer during the transient current (n = 9).
In the protocol of Figure 3, the resurgent current followed a 20 msec
pulse to +30 mV. Briefer prepulses, however, also elicited resurgent
current. Figure 5 shows the current elicited by a return to 30 mV after steps to +30 mV of 1, 2, and 5 msec. Resurgent sodium
current of similar size followed each of these pulses. With a 1 msec
step to +30 mV, inactivation was not maximal, and there was a
conventional fast-tail current that rose in ~50 µsec and decayed in
~500 µsec; this tail was followed by a much slower secondary phase
of resurgent current. With a 2 msec pulse to +30 mV, inactivation was
more complete, the tail current was smaller, and the rising phase of
the resurgent current was more pronounced. With a 5 msec pulse to +30
mV, inactivation was maximal and there was no detectable tail current.
The rising phase of the resurgent current was most evident with the 5 msec step to +30 mV because there was no conventional tail current, but
the peak size of the resurgent current was very similar with all three
pulses. A 1 msec pulse to +30 mV is sufficient, apparently, to produce
a maximal resurgent current.
Fig. 5.
Resurgent sodium current after brief steps to +30
mV. Resurgent current at 30 mV is shown after steps to +30 mV of 1, 2, and 5 msec. After the briefest steps to +30 mV, tail currents (arrows) are followed by the resurgent current. The peak
current at +30 mV (offscale) is 3100 pA.
[View Larger Version of this Image (16K GIF file)]
Does the resurgent current flow through the same population of
sodium channels that underlie the conventional transient current elicited by a simple depolarization? We examined this question with
single-channel recordings from cell-attached patches in Purkinje neurons. By using relatively small-tipped quartz pipettes (10-20 M), it was possible to obtain patches containing only a single channel with a reasonable frequency. The presence of only one channel
was determined by the absence of openings to multiples of the unitary
amplitude for at least 200 sweeps with steps from 90 mV to test
voltages from 30 to 0 mV. Because the peak open probability was
0.3-0.6 for such depolarizations, this is a stringent test for the
presence of multiple channels. Of 49 patches with sodium channel
activity where long-lived, low-noise recordings were obtained with no
interference from other types of channels, 10 patches contained a
single channel and were studied further. Figure 6 shows
the channel activity elicited in such a patch by a protocol that would
elicit resurgent current effectively in a whole-cell experiment. From a
holding voltage of 90 mV, the patch was stepped to 30 mV for 40 msec, +30 mV for 15 msec, 30 mV for 40 msec, and back to 90 mV. The
single-channel currents evoked by this series of voltage steps had an
amplitude of 1.7 pA at 30 mV. Despite the clustering of openings at
the beginning of the first step to 30 mV, openings to twice the
unitary amplitude were never observed in 1400 steps, which suggests the
presence of only one channel.
Fig. 6.
Single sodium channels in Purkinje neurons.
A, Two sets of eight consecutive traces recorded from a
one-channel patch are shown. Fourteen hundred such sweeps were recorded
from this patch. B, Latency histograms of bursts during
the first and second epochs at 30 mV.
[View Larger Version of this Image (44K GIF file)]
Although most openings occurred in the first few milliseconds of the
first step to 30 mV, there were also openings during the second epoch
at 30 mV. In fact, similar to macroscopic resurgent current, single
channels showed much more activity during the second epoch at 30 mV
than was present during the first epoch once the initial transient
current had decayed. We quantified this behavior by counting openings
after the first 4 msec of each epoch at 30 mV. Openings in both the
first epoch and second epoch occurred in well defined bursts with very
short intraburst closings, so we counted bursts rather than individual
openings, ignoring closures <70 µsec. In 1400 sweeps, in the first
epoch at 30 mV, there were 18 bursts after the first 4 msec (of a
total of 1808 bursts), whereas in the second epoch there were 74 bursts
after the first 4 msec (of 90 bursts). Figure 6B
shows histograms of the latency of all bursts during the first epoch
(top) and second epoch (bottom) at 30 mV. The
comparison shows that the bursts during the second epoch are far in
excess of what would be expected by simple incomplete inactivation or
persistent current. Rather, late openings are facilitated after the
intervening depolarization to +30 mV, just as in the macroscopic
current. In principle, the current formed by the ensemble average of
the single-channel events during the second epoch could be compared
directly with macroscopic resurgent current, but we found that 90 bursts are too few to form a well resolved ensemble current, because
the bursts are relatively brief and occur throughout the 40 msec
epoch.
Nine of the ten single-channel patches appeared to have a channel that
gave "resurgent" openings as well as transient openings, as
determined by the presence of substantially more openings during the
last 36 msec of the 30 mV pulse after the step to +30 mV than during
the same period of the first pulse to 30 mV. The tenth single channel
appeared to have "conventional" behavior, with very few openings
after the first 4 msec for the first step to 30 mV and only one burst
(in 500 sweeps) in the second epoch. In the nine patches showing
resurgent openings, the number of openings in the final 36 msec of the
second (40 msec) epoch was two- to ninefold those in the same period of
the first epoch. In all 10 patches, the depolarization and
repolarization openings had the same amplitude, consistent with both
arising from the same channel.
In 1 of the 49 patches from which stable recordings of sodium
channel activity were made, a channel showing purely noninactivating kinetics was active. This channel had normal voltage dependence of
activation, but showed no decay of channel activity during 40 msec
depolarizations to voltages from 30 to +30 mV. Such noninactivating channel activity was described previously by Sugimori et al. (1992) in
recordings from Purkinje neurons in slices, although the channel we
recorded had a conductance of 20 pS, identical to the other sodium
channels and different from the lower-conductance channel in the
recordings of Sugimori et al. Because of its infrequent appearance, we
did not study this channel type further. Such channels could well
contribute to the small persistent current in our cells but would not
be expected to contribute to the resurgent current, because they would
give steadily maintained activity during a 30 to +30 to 30 mV
sequence.
How can the resurgent current be understood in terms of channel
gating states? An interesting possibility is that the current results
from channels that recover from inactivation by passing transiently
through open states. Such a current has been demonstrated for Shaker
potassium channels by Demo and Yellen (1991). In fact, this model is
consistent with the results in Figure 5. After the 1 msec step to +30
mV, which allows partial inactivation, channels are in open and
inactivated states. Repolarization to 30 mV leads to reequilibration
between these states, giving rise to the fast tail (some closure of
open channels), peak resurgent current (some opening of inactivated
channels), and decay to a steady-state (inactivation and/or closure of
open channels). After the 5 msec step to +30 mV, channels are almost
fully inactivated, and upon repolarization, inactivated channels may
reopen to give the resurgent current. To examine this idea further, we
measured recovery from inactivation at the potentials where resurgent
current flows, as shown in Figure 7A. After a
25 msec conditioning pulse to 0 mV to produce maximal inactivation, the
membrane was held at 40 mV for various intervals, and recovery was
assessed with a test pulse to 0 mV. After 15 msec at 40 mV, currents
elicited by test pulses had recovered to a steady-state level of
~11% of the peak transient current evoked by the conditioning pulse;
this is consistent with the average value of 10 ± 6%
(n = 8) availability determined from inactivation
curves. The time course of recovery at 40 mV could be fit well by a
single exponential of ~4 msec (dashed line, Fig.
7B). Resurgent current flowed during the recovery period at
40 mV, consistent with the idea that the current is associated with
recovery.
Fig. 7.
Recovery from inactivation at 40 mV in Purkinje
and CA3 neurons. A, Responses to the voltage protocol
shown for a Purkinje cell (top traces) and CA3 cell
(bottom traces). The initial conditioning step from 90
to 0 mV was 25 msec. Intervals at 40 mV were 1-14.5 msec in 1.5 msec
increments. B, Amplitudes of recovery currents were
normalized to the peak current in response to the conditioning step,
and the percentage of recovery is plotted against interval. Data were
fitted with a single exponential function,
where %max is the maximal recovery, t
is the interval (msec), is the time constant of recovery, and
%max + A is the noninactivated percentage of
current (0.77% for this cell).
[View Larger Version of this Image (13K GIF file)]
In contrast to Purkinje neurons, in CA3 neurons there was no rapid
phase of recovery at 40 mV and no resurgent current (Fig. 7C). There was some recovery from inactivation with long
times at 40 mV (consistent with steady-state inactivation at 40 mV, leaving ~7% availability, as determined from inactivation curves), but it was so slow that there was <1% recovery in 15 msec. The lack
of resurgent current in CA3 neurons is consistent with a previous study
of CA1 neurons in which current associated with recovery from
inactivation was not detected (Kuo and Bean, 1994). In that study,
recovery seemed to become slower monotonically at less negative
voltages and had a time constant of 20 msec at 70 mV. Thus, it seems
that recovery at 40 mV in hippocampal neurons is far slower than in
Purkinje neurons, even though the steady-state inactivation at 40 mV
in CA3 neurons (7 ± 6%, n = 4) is not
dramatically different from that in Purkinje neurons (10 ± 6%,
n = 8).
Comparison of the CA3 cell and Purkinje cell data suggests a
correlation between the existence of resurgent current and recovery from inactivation at moderately negative voltages. A plausible explanation is that at moderately negative potentials the predominant sodium channels in Purkinje cells can recover partially from
inactivation by passing transiently through an open state.
It seems likely that the resurgent current is important
physiologically. It is largest at voltages near the threshold for action potential formation (50 to 40 mV), where the cell is likely
to be most sensitive to small currents. The brief steps to +30 mV that
are sufficient to trigger the resurgent current in Figure 4 are similar
to the voltage change expected during a fast action potential. To
investigate directly whether the resurgent current may be activated
after action potentials, we recorded action potentials under current
clamp and then used them as command voltages in voltage-clamp
recordings.
We expected that the action potentials in dissociated cells might
be considerably different from those in intact neurons, as a result of
loss of the dendritic tree and possible loss of resting potential
during the dissociation. To our surprise, the properties of the
dissociated cells under current clamp turned out to be remarkably
similar to intact neurons in cerebellar slices or in tissue culture. In
particular, the dissociated neurons showed two of the distinctive
properties of Purkinje neurons in vivo and in slices:
spontaneous firing and a tendency to fire multipeaked action
potentials. Figure 8A shows the
typical behavior of a dissociated Purkinje neuron under
quasiphysiological ionic conditions with no current injected into the
neuron. The cell fired spontaneous action potentials with an amplitude
of 80 mV (62 to +18 mV) at a highly regular rate (every 35 msec).
Spontaneous firing was seen in six of six neurons examined. The
spontaneous firing was not caused by whole-cell recording, because
cells fired at a similar rate before being dialyzed, as detected by
action currents when the pipette was sealed onto the cell. The rate of
firing was speeded by injection of depolarizing current and was stopped
by injection of a hyperpolarizing current. The cells fired until the
steady holding current was adjusted to reach membrane potentials near 80 mV.
Fig. 8.
Spontaneous firing and conglomerate action
potentials in acutely isolated Purkinje cells. A,
Spontaneous action potentials recorded in an isolated Purkinje cell.
B, Voltage responses of a different Purkinje cell in
response to a 1 msec current injection of 1.2 and 1.4 nA. The smaller
injection produced the subthreshold response. A steady injection of
40 pA brought the resting potential to 85 mV and stopped the
spontaneous firing. Note different time scale from
A.
[View Larger Version of this Image (18K GIF file)]
Figure 8B shows records from a cell in which
the spontaneous firing was stopped by a steady holding current of 40
pA, which brought the membrane potential to 85 mV. We then stimulated
action potentials by injecting depolarizing current. To avoid injected current overlapping with the action potential, we injected large currents for a brief time (1 msec), mimicking the "shock" protocol used by Hodgkin and Huxley (1952) to stimulate membrane action potentials. The brief, large depolarizations also mimic synaptic currents. The cell fired with a sharp voltage threshold of
approximately 55 mV. Even with stimulation that was just
suprathreshold, the cell fired a burst of three action potentials.
All-or-none firing of a burst of action potentials was seen in six of
six cells. In five cells, the burst had three spikes, whereas in the
sixth there was an all-or-none quadruple action potential. The final spike was always followed by an afterdepolarization (arrow,
Fig. 8B). The action potential formation in
dissociated Purkinje neurons is different from most other cell types,
where injection of a short pulse of current elicits a single spike and
where afterhyperpolarization is much more common than
afterdepolarization. For simplicity, we will refer to the all-or-none
burst elicited by a brief current injection as a "conglomerate"
action potential.
To determine if resurgent sodium current was activated during the
conglomerate action potential waveform, we used the waveform as command
voltage in a voltage-clamp experiment in which sodium current was
isolated by appropriate solutions. The result of this experiment is
shown in Figure 9. There was a large transient current (1.7 nA) at the time of the upstroke of the first spike. Substantial sodium current continued to flow after the first spike. In the late
phase of the slow depolarization leading up to the second spike, the
sodium current reached ~200 pA. This sodium current flowing between
spikes probably corresponds mainly to resurgent current rather than
persistent current, because this cell had only small persistent current
at the end of a conventional step depolarization (66 pA at 30 mV).
When the same conglomerate action potential waveform was applied to CA3
neurons (Fig. 9B), there was much less sodium current
elicited after the initial transient associated with the upstroke of
the first action potential.
Fig. 9.
Sodium currents evoked by conglomerate
action potential waveforms in Purkinje and CA3 neurons.
A, The top panel illustrates the command
potential (Vcmd) applied to a
voltage-clamped Purkinje cell. The command waveform was obtained from a
conglomerate action potential. Thin horizontal lines
indicate 0, 45, and 75 mV, on Vcmd as
labeled. The middle panel (heavy line)
shows the Na+ current (in 50 mM
Na+) evoked in this cell by the voltage protocol. The
bottom panel shows the total current flux in
physiological solution (140 Na+ plus K+ and
Ca2+; see Materials and Methods) calculated from the
product of dV/dt of the spike waveform
and the cell capacitance Cm of the cell in
which the conglomerate action potential was recorded. The total current
is plotted on the same scale as the
INa+ to facilitate comparison. The peak
Itotal is 3525 pA and is offscale in the
figure. Dashed lines at 0 pA are included on all the
current traces. Vertical dotted lines indicate the
relative amplitudes of INa+ and
Itotal at the onset of the second and
third action potentials. B, Same voltage protocol as
A applied to a CA3 cell.
[View Larger Version of this Image (23K GIF file)]
It is possible to compare the size of the sodium current elicited
by the conglomerate action potential with the flow of the overall ionic
current. Because the membrane during an action potential in an isolated
cell body is isopotential, we can directly derive the total ionic
current flowing during the action potential as CmdV/dt, where
Cm is the membrane capacitance and
dV/dt is the first derivative of the voltage.
Because total membrane current is the sum of capacitative current
(CmdV/dt) and ionic
current, when the total membrane current is zero (as it is after the
short current injection is off), Iionic = CmdV/dt (Hodgkin and
Huxley, 1952). The bottom trace in Figure 9A
shows CmdV/dt calculated from the action potential, using the average value of 25 pF for Cm. Clearly, the magnitude of sodium current
flowing during interspike intervals is substantial (even when recorded
with 50 mM Na+) compared with the net ionic
current (in full Na+). This suggests that, whatever other
ionic currents may be flowing, the sodium current exerts a major
influence on the slow depolarizations leading up to the second and
third spikes.
If the current between spikes in the Purkinje neurons corresponds
to resurgent current, it may be associated with partial recovery from
inactivation. We therefore examined the extent of inactivation after
the first spike of the conglomerate action potential and tested whether
there was recovery from inactivation between the first and second
spike. The voltage protocol was essentially a conventional double pulse
test of recovery from inactivation, but using the first spike and
following trough of a conglomerate action potential as the prepulse and
the recovery interval (Fig. 10A). The
extent of recovery was tested by a test pulse to 0 mV (preceded by 0.5 msec at 100 mV to ensure activation of available channels). The
resulting currents are shown in Figure 10B.
Inactivation and recovery are reflected in the envelope of test current
peaks and are plotted in the inset. The test currents were normalized to the maximal current evoked by a step directly to 0 mV from the
initial holding potential of 75 mV (Fig. 10C). The number of available channels continues to decline during the falling phase of
the first spike. But when the cell membrane potential reaches the
trough between the first and second spikes, recovery begins. Before the
second spike, ~20% of the channels are available for activation.
Again, there is a striking contrast in the behavior of CA3 neurons,
which show very little recovery in this period (open
triangles, inset, Fig. 10B).
Fig. 10.
Inactivation and recovery of Na+
current during the conglomerate action potential. A,
Responses of a Purkinje cell evoked by the voltage protocol shown.
After increasing durations of the action potential waveform, test
current was elicited at 0 mV (after a 0.5-msec step to 100 mV).
Durations of the action potential waveform (and voltages reached) were
2.85 msec (10 mV), 3.2 msec (10 mV, bold traces), 3.65 msec (30 mV), 4.95 msec (46 mV), 6.35 msec (43 mV), and 7.35 msec
(39 mV). The inset to A plots the
percentage of recovery on the same time base as the traces, for the
Purkinje cell shown (filled symbols), and for a
representative CA3 cell (open symbols). Percentage of
recovery was calculated as the peak test current at 0 mV normalized to
the maximal current evoked by a step to 0 directly from the 75 mV
holding potential. The amount of recovery that occurs during a half
msec at 100 mV, ~7%, is reflected by the dotted
line (see traces in B).
B, The response of the Purkinje cell in A
to the step protocol shown.
[View Larger Version of this Image (17K GIF file)]
DISCUSSION
Our results show unusual behavior of sodium current in cerebellar
Purkinje neurons: after strong depolarizations, returning the membrane
to voltages in the range of 60 to 20 mV elicits resurgent current.
This behavior is not expected of "conventional" sodium channels.
For conventional channels, a step from +30 mV to 30 mV would not
elicit current, because channels would be maximally inactivated at +30
mV and would remain inactivated and nonconducting at 30 mV. Indeed,
no such current was seen in CA3 neurons using this protocol.
The channels underlying the resurgent sodium current are
TTX-sensitive: all of the currents shown in this paper were obtained by
TTX-subtraction, and no detectable voltage-dependent current remained
in the presence of TTX. The single-channel experiments show that the
same channels that produce resurgent current also produce transient
current for a simple depolarization from rest so that, in principle,
all of the sodium current could arise from a single channel type.
However, 1 of 10 single channel patches seemed to possess a
conventional sodium channel that produced transient current but not
extra, resurgent openings, suggesting that channels capable of
producing resurgent openings might coexist with conventional channels.
The channels sampled in patches that were selected because they
contained only a single channel do not necessarily constitute a random
sample of the channels underlying the macroscopic current in Purkinje
neuron, because there may be clusters of channels. Thus, it is possible
that there is more heterogeneity in sodium channels underlying the
macroscopic current than is suggested by the single-channel results.
There may also be heterogeneity in the membrane that contributes the
macroscopic current. The membrane of the dissociated cells is primarily
from the soma, but there is also a stump remaining from the proximal dendrite (Regan, 1991). In addition, there may be membrane from the
axon hillock, which may possess a high density of sodium channels.
At a mechanistic level, the resurgent current may represent
recovery from inactivation proceeding through open states of the channel. The evidence for this, however, is indirect and
circumstantial: that the resurgent current in Purkinje neurons is
accompanied by partial, rapid recovery from inactivation and that the
sodium current in CA3 neurons has neither resurgent current nor rapid recovery from inactivation at 40 mV. Tail currents that may
correspond to recovery through open states of sodium channels have been
reported previously for TTX-resistant sodium channels in sensory
neurons (Rizzo et al., 1994), although the lack of sensitivity to TTX made unequivocal identification of the permeant ion difficult. The late
tail currents recorded by Rizzo and colleagues had much slower decay
kinetics than the resurgent current in Purkinje neurons, because they
were evident at voltages of 100 to 140 mV, where the resurgent
current decays too quickly to be resolved. If the basic mechanism
underlying these currents in sensory neurons is similar to the
mechanism in Purkinje neurons, it will interesting to see if the
current is correlated with a tendency to fire repetitively in the
subset of sensory neurons showing the behavior.
It seems very likely that the unusual properties of the channels
underlying the resurgent sodium current are related closely to the
distinctive firing behavior of Purkinje neurons, especially the ability
to fire multipeaked action potentials. As shown directly in Figure 9,
the sodium current that flows after one spike is enough to contribute
significantly to the afterdepolarization leading up to the second spike
in the conglomerate action potential. Of course, changes in other ionic
currents are also occurring during this time and must influence firing
of the second spike. In particular, the large P-type calcium currents
present in Purkinje neurons would be active at these voltages of 50
to 30 mV and would be inactivated only partially (Regan, 1991).
Calcium entry through calcium channels, however, may well activate
counterbalancing potassium currents through calcium-activated potassium
channels (Cardozo and Bean, 1995). Whatever other ionic currents flow
between the first and spikes of the action potential, the sodium
current flowing during this time is a substantial part of the net
current, as obtained from
CmdV/dt.
In cerebellar slices, intact Purkinje neurons fire complex spikes with
climbing fiber stimulation (Eccles et al., 1966; Llinás and
Sugimori, 1980a) but not parallel fiber stimulation or current injection (Stuart and Häusser, 1994), whereas with dissociated neurons we find all-or-none firing of multiple action potentials even
with brief current injections. The factors underlying the differences
in firing of intact and dissociated Purkinje neurons remain to be
determined. Our results suggest that resurgent sodium current in the
cell body confers an intrinsic tendency to repetitive firing, but
firing patterns in intact neurons will also be influenced strongly by
dendritic conductances and synaptic currents. In particular, the
complex spikes elicited in intact cells by climbing fiber stimulation
are shaped undoubtedly by the prolonged synaptic potential and by
dendritic conductances (Fujita, 1967; Llinás and Sugimori, 1980b)
as well as by voltage-dependent current in the cell body.
The comparison between Purkinje and CA3 neurons suggests that the
resurgent current is associated with unusually rapid (though partial)
recovery from inactivation at voltages near 40 mV. The partial
recovery from inactivation at relatively positive voltages is likely to
be an important factor in determining the ability to fire conglomerate
action potentials. With conventional sodium channels, as in CA3
neurons, there is essentially no recovery from inactivation in 10-15
msec at 40 mV so that the cell would be truly refractory during an
afterdepolarization to this voltage. In the Purkinje neuron, the rapid
recovery from inactivation of TTX-sensitive current at such voltages
would make channels available to give rise to a second spike. Even
availability of only 20% of the maximal sodium current in the
interspike interval (Fig. 10) is likely to be more than enough for
generation of a robust second spike.
The unusual gating properties of TTX-sensitive sodium current in
Purkinje neurons illustrate the extent to which the voltage-dependent channels underlying action potentials in mammalian central neurons can
differ from those in the squid axon (Llinás, 1988). Even the
qualitative behavior of sodium channels in Purkinje neurons differs
from that in other cell types, and the activation of resurgent current
is likely to influence significantly the firing pattern of the cell.
Similar adaptations may be present in sodium channels of other neurons
that exhibit repetitive or oscillatory firing, behavior common to a
variety of central neurons.
FOOTNOTES
Received Jan. 30, 1997; revised March 22, 1997; accepted March 27, 1997.
This work was supported by National Institutes of Health Grant HL35034.
We are grateful to Dr. N. V. Marrion for advice on single-channel
recording and Drs. J. T. Williams and D. Bergles for guidance on
current-clamp recording.
Correspondence should be addressed to Dr. Indira M. Raman, Department
of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston,
MA 02115.
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