Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5513
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ABSTRACT |
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Pan, Enhui and Costa M. Colbert. Subthreshold Inactivation of Na+ and K+ Channels Supports Activity-Dependent Enhancement of Back-Propagating Action Potentials in Hippocampal CA1. J. Neurophysiol. 85: 1013-1016, 2001. Back-propagating action potentials in CA1 pyramidal neurons may provide the postsynaptic dendritic depolarization necessary for the induction of long-term synaptic plasticity. The amplitudes of back-propagating action potentials are not all or none but are limited in amplitude by dendritic A-type K+ channels. Previous studies of back-propagating action potentials have suggested that prior depolarization of the dendritic membrane reduces A-type channel availability through inactivation, resulting in an enhanced, or boosted, dendritic action potential. However, inactivation kinetics in the subthreshold potential range have not been directly measured. Furthermore, the corresponding rates of Na+ channel inactivation with depolarization have not been considered. Here we report in cell-attached patches (150-220 µm from the soma, 32°C) that at 20-mV positive to rest, A-type K+ channels inactivated with a single exponential time constant of 6 ms, whereas Na+ channels inactivated with a time constant of 37 ms. The ratio of available Na+ to K+ current increased as the duration of the depolarization increased. Thus the subthreshold properties of Na+ and A-type K+ channels provide a mechanism by which information about the level of synaptic activity may be encoded in the amplitude of back-propagating action potentials.
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INTRODUCTION |
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Back-propagating action
potentials in CA1 pyramidal neurons may provide the postsynaptic
dendritic depolarization necessary for the induction of associative
long-term synaptic plasticity (Hoffman et al. 1997;
Magee and Johnston 1997
). Unlike somatic or axonal
action potentials, back-propagating dendritic action potentials are not
all-or-none but are highly variable in amplitude (Colbert et al.
1997
; Spruston et al. 1995
). This variable
amplitude is determined in large part by a high density of dendritic
A-type K+ (Kv4.2) channels (Colbert and
Pan 1999
; Hoffman et al. 1997
), which are the
targets of various neuromodulatory systems (Hoffman and Johnston
1999
), and subject to voltage-dependent inactivation (Hoffman and Johnston 1999
; Hoffman et al.
1997
).
Depolarization of the dendritic membrane by prior synaptic
activity can enhance the amplitude of back-propagating dendritic action
potentials (Hoffman et al. 1997) (Fig.
1), which increases the likelihood of
Ca2+ entry through voltage-gated and
N-methyl-D-aspartate channels (Hoffman et
al. 1997
; Magee and Johnston 1997
). Thus
enhancement of the back-propagating action potential may contribute to
the associative nature and the specific timing requirements for the induction of long-term synaptic modification (Bi and Poo
1998
; Debanne et al. 1998
;
Hashemzadeh-Gargari et al. 1991
; Levy and Steward
1983
; Markram et al. 1997
). Because of the
important role of A-type K+ channels in shaping
the dendritic action potential, it has been hypothesized that rapid
inactivation of these channels during depolarization underlies the
enhancement of dendritic action potential amplitude (Hoffman et
al. 1997
; Migliore et al. 1999
).
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Certain aspects of this putative mechanism, however, have not been
directly investigated. First, the rate of subthreshold Na+ channel inactivation has not been determined.
Second, A-type K+ channels inactivate rapidly in
the supra-threshold range (Hoffman et al. 1997), but
subthreshold inactivation rates have only been extrapolated. Finally,
the various kinetics have not been measured at temperatures above room
temperature. Thus to test whether inactivations of
Na+ and K+ channels are
consistent with enhancement of back-propagating action potentials, we
recorded currents in cell-attached patches from dendrites of CA1
pyramidal neurons. We found that both the rates and degree of
inactivation with near-threshold synaptic depolarization led to an
increase in the ratio of inward to outward currents consistent with an
enhancement of action potential amplitude.
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METHODS |
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Slice methods are essentially those described in Colbert
and Pan (1999). Briefly, the experiments used 4-to 6-wk-old
male Sprague- Dawley rats. Animals were deeply anesthetized with a combination of ketamine and xylazine and perfused through the heart
with cold artificial cerebrospinal fluid (ACSF) containing (in mM) 110 sucrose, 60 NaCl, 3.0 KCl, 1.25 NaH2PO4, 28 NaHCO3, 0.5 CaCl2, 7.0 MgCl2, and 5 dextrose. Slices 400-µm thick were cut using a Vibratome (Lancer).
The external ACSF contained (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2.0 CaCl2, 1.0 MgCl2, and 25 dextrose. Slices were maintained submerged in normal ACSF bubbled continuously with 95% O2-5% CO2. The pipette solution used for whole cell recordings contained (in mM) 140 K-Gluconate, 10 HEPES, 1 EGTA, 4.0 NaCl, 4.0 Mg2ATP, 0.3 Mg2GTP, and 14 phosphocreatine; pH was 7.25. The pipette solution for cell-attached patch recordings of Na+ currents contained (in mM) 110 NaCl, 20 tetraethylammonium (TEA) chloride, 10 HEPES, 2 CaCl2, 3 KCl, 1 MgCl2, and 10-12 4-aminopyridine (4-AP). pH was adjusted to 7.4 with NaOH. For cell-attached recordings of Na+ and A-type K+ currents, 4-AP was replaced by NaCl (5 mM). For cell-attached recordings of A-type currents alone, 4-AP was replaced by NaCl (5 mM) and tetrodotoxin (TTX, 1 µM) was added.
Dendrites of CA1 pyramidal neurons were visualized using
infrared-illuminated, differential interference contrast optics
(BX50WI, Olympus) and a newvicon camera (DAGE-MTI) according to
standard techniques (Stuart et al. 1993). Whole cell
patch-clamp recordings in the apical dendrites were made using an
intracellular amplifier (BVC-700, Dagan). Cell-attached patch
recordings were made using a patch-clamp amplifier (Axopatch 200, Axon
Instruments). Pipettes (5-7 M
for whole cell, 7-10 M
for
cell-attached) were made from EN-1 glass (Garner) and pulled using a
P-97 Flaming-Brown pipette puller (Sutter Instruments). All recordings
were made at 32 ± 0.5°C, mean ± SD. Membrane
potentials were determined by rupturing patches at the end of the
recording sessions and were about
70 mV, consistent with other
studies of CA1 dendrites (Hoffman et al. 1997
;
Magee 1998
). However, data were most often collected until loss of the patch, precluding an estimate of membrane potential. Thus command potentials are reported here as depolarization from rest.
Conditioning pulse durations were interleaved to minimize any effects
of systematic changes in potential during an experiment. Ensemble
waveforms were constructed from 15-25 individual sweeps. Whole cell
recordings were low-pass filtered at 3 kHz (6 dB/octave) and digitized
at 10 kHz. Cell-attached patch recordings were filtered at 2 kHz
(8-pole Bessel filter) and sampled at 10 kHz. Data were digitized at
16-bit resolution (ITC18, Instrutech) and stored by computer (Intel)
for off-line analysis. Excitatory postsynaptic potentials (EPSPs) and
antidromic action potentials were evoked by constant current pulses
[0.1 ms, 30-100 µA (Neurolog, Digitimer)] through tungsten
electrodes (AM Systems) placed in stratum radiatum and the alveus,
respectively. Exponential curve fits were made with DISCRETE
(Provencher 1976
). Summary data are reported as means ± SE.
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RESULTS |
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Back-propagating action potentials in CA1 pyramidal neurons decrease in amplitude as they travel through the apical dendrites. By 150-200 µm, they reach an amplitude of 20-30 mV (Fig. 1, Anti). If paired with EPSPs, the amplitude of back-propagating action potentials is enhanced (Fig. 1A, EPSPs + Anti). The magnitude of this enhancement increases as the duration of depolarization increases prior to the initiation of the action potential (Fig. 1B). In this example, the enhancement could also be seen as an increase in the rate of rise (dV/dt, Fig. 1C).
A putative mechanism for the enhancement of the back-propagating action
potential by prior depolarization is the loss of available A-type
K+ current through inactivation. For such a
mechanism to work, K+ current must inactivate
rapidly and must inactivate more than Na+
current. We have shown previously that loss of
Na+ current reduces dendritic action potential
amplitude (Colbert et al. 1997). To compare subthreshold
inactivation of the channels, we recorded currents in cell-attached
patches in the apical dendrites 150-220 µm from the cell body. To
induce inactivation, patches were conditioned by applying a command
voltage 20-mV positive rest for durations of 5-20 ms (Fig.
2A). Conditioning intervals were followed by a depolarizing command to 60-mV positive to rest (i.e., to a potential of about
10 mV) to assess available current. A-type K+ currents inactivated rapidly and
markedly as shown in patches with A-type K+
current alone (Fig. 2B) and in patches with both
Na+ and A-type K+ currents
(Fig. 2C). In patches where both currents were present, A-type K+ channels inactivated with a single
exponential time constant of 6 ms (Fig. 2D). After 20 ms of
conditioning, only 29 ± 5% (n = 7) of the
current at rest remained available. In the same patches after 20 ms of
conditioning, 75 ± 12% (n = 7) of
Na+ current at rest remained available. In some
patches, a potential 10 mV above rest was also tested. The A-type
K+ current inactivated with a time constant of 5 ms to a value of 44 ± 5% (n = 3) of its resting
value.
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To further characterize the time course of inactivation of Na+ current, we recorded Na+ currents in additional patches using longer durations of conditioning depolarization (40-400 ms, Fig. 3A). Voltage commands to 20-mV positive to rest inactivated Na+ channels as shown by representative ensemble averages (Fig. 3B) and by summary data (Fig. 3C). Na+ current inactivated with a rate of 37 ms (single-exponential time constant). At 440 ms, 50 ± 9% (n = 5) of the Na+ current at rest remained available.
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DISCUSSION |
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In the present study, we have investigated subthreshold inactivation of ion channels underlying the back-propagating action potential. Given the surprising result that the K+ channels inactivated so much faster than the Na+ channels, the key experiment here was to compare Na+ and K+ channel inactivation in the same patches directly. Thus any errors in our estimates of membrane potential, temperature, or differences in pipette solutions can be ruled out.
Previously inactivation rates for A-type K+
channels were estimated by evoking currents and directly measuring the
rate at which the current decreased (Hoffman et al.
1997). Such measurements were necessarily limited to
supra-threshold potentials (at least
25 mV), because the currents
evoked at subthreshold potentials were too small to measure accurately.
From the observation that the rate of inactivation of A-type
K+ channels was faster with less depolarization,
it was concluded that subthreshold inactivation should proceed rapidly.
The present results provide direct support for this idea.
The very slow rate of Na+ channel inactivation
was somewhat of a surprise. In an earlier study of CA1 dendrites using
depolarizations to 40 mV to evoke currents, Magee and Johnston
(1995)
found that Na+ current
inactivated with two distinct rate constants. The fast exponential rate
constant was in the 1- to 4-ms range, the value also reported by
Sah et al. (1988)
. The slower time constant, however,
was similar to that seen in the present study (30-50 ms). Thus entry
into the inactivated state may take one of two distinct pathways. The
modest depolarizations used in the present study (which evoked little
current) might favor inactivation by the slower pathway resulting in a
large fraction of the resting current being available for up to 100 ms
after depolarization begins.
Back-propagating action potentials in the dendrites of CA1 pyramidal
neurons have been hypothesized to be a rapid signal to the synapses
that the postsynaptic cell has fired (Hoffman et al.
1997; Magee and Johnston 1997
). Such a mechanism
would seem to mirror long range signaling in the axon. However, the
amplitude of dendritic action potentials is highly variable, allowing
additional information, such as modulation by neurotransmitter systems
(Hoffman and Johnston 1999
) or the inactivation studied
here, to be integrated into the signal. One well-established property
of long-term synaptic plasticity in many systems is that induction has
specific timing requirements (Bi and Poo 1998
;
Hashemzadeh-Gargari et al. 1991
; Levy and Steward
1979
, 1983
; Markram et al. 1997
). In particular, synaptic activity following the strong input (i.e., that responsible for the postsynaptic depolarization) does not induce potentiation. Such
restricted timing seems more consistent with the brief, strong depolarization of an action potential than with the more prolonged depolarization associated with strong excitatory synaptic input. Consistent with this idea, both back-propagating dendritic action potentials and activation of NMDA receptors were required for induction
in a study of long-term potentiation (Magee and Johnston 1997
). If, under physiological conditions, a back-propagating action potential is necessary to relieve the Mg2+
block of the NMDA receptor (Nowak et al. 1984
), then it
is critical to maintain action potential amplitude in the face of
dendritic depolarization. Furthermore if a back-propagating action
potential with enhanced amplitude is necessary to activate NMDA
receptors, then synaptic modification will only progress if an adequate
number of synapses are activated in the period before an action
potential is initiated.
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ACKNOWLEDGMENTS |
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We thank J. Stringer for reading an earlier version of the manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-36982 and Texas Advanced Research Program Grant 003652-0146-1999.
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FOOTNOTES |
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Address for reprint requests: C. M. Colbert, Dept. of Biology and Biochemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204-5513 (E-mail: ccolbert{at}uh.edu).
Received 31 August 2000; accepted in final form 16 October 2000.
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REFERENCES |
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