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The Journal of Neuroscience, February 1, 1998, 18(3):854-867
Sodium Current in Rat and Cat Thalamocortical Neurons: Role of a
Non-Inactivating Component in Tonic and Burst Firing
H. Rheinallt
Parri and
Vincenzo
Crunelli
Physiology Unit, School of Molecular and Medical Biosciences,
University of Wales Cardiff, Cardiff CF1 3US, United Kingdom
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ABSTRACT |
The properties of the Na+ current present in
thalamocortical neurons of the dorsal lateral geniculate nucleus were
investigated in dissociated neonate rat and cat neurons and in neurons
from slices of neonate and adult rats using patch and sharp electrode recordings.
The steady-state activation and inactivation of the transient
Na+ current (INa)
was well fitted with a Boltzmann curve (voltage of half-maximal
activation and inactivation, V1/2,
29.84 mV and 70.04 mV, respectively). Steady-state activation and
inactivation curves showed a small region of overlap, indicating the
occurrence of a INa window current.
INa decay could be fitted with a single exponential function, consistent with the presence of only one channel
type.
Voltage ramp and step protocols showed the presence of a
noninactivating component of the Na+ current
(INaP) that activated at potentials
more negative (V1/2 = 56.93 mV) than those
of INa. The maximal amplitude of
INaP was ~2.5% of
INa, thus significantly greater than
the calculated contribution (0.2%) of the
INa window component. Comparison of results
from dissociated neurons and neurons in slices suggested a dendritic as
well as a somatic localization of INaP.
Inclusion of papain in the patch electrode removed the fast
inactivation of INa and induced a current
with voltage-dependence (V1/2 = 56.92) and
activation parameters similar to those of
INaP.
Current-clamp recordings with sharp electrodes showed that
INaP contributed to depolarizations evoked
from potentials of approximately 60 mV and unexpectedly to the
amplitude and latency of low-threshold Ca2+
potentials, suggesting that this noninactivating component of the
Na+ channel population plays an important role in
the integrative properties of thalamocortical neurons during both tonic
and burst-firing patterns.
Key words:
thalamus; action potential; persistent
Na+ current; inactivation; burst firing; dorsal
lateral geniculate nucleus
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INTRODUCTION |
The thalamocortical loop has been
the subject of many electrophysiological studies because of its central
role in awareness and sleep (Ribary et al., 1991
; Steriade et al.,
1993; Sillito et al., 1994; Barth and MacDonald, 1996) as well as in a
number of neurological disorders (Malafosse et al., 1994; Jeanmonod et al., 1996). In particular, the ionic currents of its constituent neurons have been investigated extensively to determine their contribution to different electrophysiological behaviors (Steriade et
at., 1990; Huguenard and Prince, 1991; Llinás et al., 1991; Connors, 1994; McCormick and Bal, 1997). A surprisingly noticeable exception, however, has been the transient Na+
current (INa). Although thoroughly
characterized in cortical pyramidal neurons during both developmental
and pathological states (Huguenard et al., 1988; Fleidervish et al.,
1996), there has been no study aimed at investigating the biophysical
properties of this current in, and its precise contribution to the
tonic and burst firing of, thalamocortical (TC) neurons. Thus,
biophysical modeling of TC neurons (McCormick and Huguenard, 1992;
Tóth and Crunelli, 1992; Destexhe et al., 1993; Antal et al.,
1996) has relied on the modified description of Na+
currents from squid axons and sympathetic, neocortical, and hippocampal neurons (French et al., 1990; Belluzzi and Sacchi, 1991; Traub and
Miles, 1991; Traub et al., 1991), a less than optimal compromise in
view of the existence of voltage-dependent Na+
channel isoforms with different biophysics, pharmacology, and tissue
distribution (Noda et al., 1986; Heinemann et al., 1992; Roy and
Narahashi, 1992).
Recently, a renewed interest in the noninactivating
Na+ current INaP has
developed. This sustained component of the Na+
current is seen in many excitable cells, including central neurons (French et al., 1990; Alzheimer et al., 1993; Crill, 1996; Fleidervish and Gutnick, 1996), and it has been implicated in signal amplification (Stuart and Sakmann, 1995; Lipowsky et al., 1996) and intrinsic high
frequency oscillations of neocortical neurons (Llinás et al.,
1991; Silva et al., 1991). The mechanism or identity of
INaP is still a matter of some controversy, with
multiple theories being advanced in different systems (Alzheimer et
al., 1993; Sugimori et al., 1994; Crill, 1996). Indirect evidence for
the presence of INaP in mammalian TC neurons is
limited to the effect of TTX in current-clamp recordings (Jahnsen and
Llinás, 1984b; Tennigkeit et al., 1996; Pedroarena and
Llinás, 1997). A description of the steady-state and kinetic
properties of INaP in these neurons would be
significant for a fuller understanding of their signal integration
properties in physiological functions and neurological conditions and
would also provide the necessary data for ongoing simulation studies of
the activity of single and small networks of thalamic neurons.
In this study we have determined the properties of
INa and INaP in TC
neurons using patch-clamp recordings in the dorsal lateral geniculate
nucleus (dLGN) of neonate rats and cats and adult rats, and we have
investigated the physiological role of INaP in
tonic and burst firing using current-clamp microelectrode recordings in
adult rats.
A preliminary report of some of these results has been published
previously (Parri et al., 1996).
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MATERIALS AND METHODS |
Preparations
Neonate rat. Slices containing the dLGN were obtained
from male Wistar rats as described by Leresche (1992). Briefly, 7- to 11-d-old rats were anesthetized with halothane (2%) and decapitated. The brain was removed, a block of tissue containing the dLGN was separated from the rest of the brain, and 350-µm-thick slices were
prepared from this tissue block using a vibratome (Energy Beam
Science). Dissection and slicing procedures were performed in ice-cold
medium of the following composition (in mM): NaCl 120, KCl
2, MgCl2 4, PIPES 20, CaCl2 1, glucose 25, ascorbic acid 0.3, kynurenic acid 1, pH 7.35 with NaOH. All chemicals
were obtained from Sigma (St. Louis, MO) unless stated otherwise.
Slices were stored in an oxygenated (100% O2)
storage bath until recording commenced. After at least 1 hr, one slice
was then anchored in the recording chamber by use of nylon threads
fixed across a platinum harp and perfused continuously with the
required recording solution (see below).
Dissociated neurons were prepared according to the procedure described
by Hernandez-Cruz and Pape (1989), Budde et al. (1992), and Oh et al.
(1995). Briefly, slices from neonate rats were prepared as detailed
above and treated with 3 mg/ml protease XXIII for 25-40 min in a glass
chamber of design similar to that described by Kay and Wong (1986).
Neurons were triturated in Ca2+-free Ringer's
solution using fire-polished Pasteur pipettes of decreasing tip
diameter. Dissociated neurons were then plated onto coverslips coated
with 1 mg/ml poly-D-lysine and allowed to settle for ~5
min before recording commenced.
Adult rat. Slices were prepared as described previously
(Crunelli et al., 1987; Williams et al., 1996). Briefly, male Wistar rats (150-200 gm) were anesthetized (2% halothane) and decapitated. A
block of tissue containing the thalamus was dissected, and
400-µm-thick slices containing the dLGN were cut using a vibroslicer
(Campden Instruments). All dissection and slicing procedures were
performed in ice-cold medium of the following composition (in
mM): NaCl 134, NaHCO3 16, KCl 5, KH2PO4 1.25, MgSO4 5, CaCl2 2, and glucose 10. Slices were maintained at room
temperature in this Ringer's solution and bubbled with a 95%
O2, 5% CO2 mixture.
Neonate cat. Cats (7-10 d old) were anesthetized (1%
halothane, 2% N2O), and the brain was removed as described
previously (Pirchio et al., 1997). From a block of tissue containing
the dLGN, 400-µm-thick slices were prepared in ice-cold medium (see above) using a Campden vibroslicer, and dissociated neurons were then
produced using the same methods as described above for the neonate
rat.
Electrophysiology
Patch-clamp recordings of identified neurons. The
slice or the coverslip containing the dissociated neurons was placed in a recording chamber mounted on the stage of a Nikon Axiophot
microscope. TC neurons were identified, and were distinguishable from
interneurons, by their characteristic size (soma diameter: 20-30 µm)
and multipolar morphology (Hernandez-Cruz and Pape, 1989; Leresche,
1992; Williams et al., 1996). Membrane currents were recorded at room
temperature (18-22°C) (except those from adult rat slices, see
below) using an Axopatch 200A (Axon Instruments, Foster City, CA).
Patch electrodes were pulled from borosilicate glass (GC120F, Clark
Electromedical Instruments, Pangbourne, UK) using a horizontal
electrode puller (Sutter Instruments, Novato, CA) and had resistances
of 1-4 M
when filled with CsF internal solution. Electrodes were
coated with Sylgard (Corning, Corning, NY) to counteract electrode
capacitance artifacts. Series resistances (4-10 M) were compensated
(60-80%) using the compensatory circuits of the amplifier, and data
were not used for analysis if the calculated maximum uncompensated error was 
5 mV. Voltage protocols, data acquisition, and analysis were controlled with pClamp (Axon Instruments). Currents were sampled
at 40 kHz and filtered with a low-pass Bessel filter at 5 kHz. Membrane
capacitance was measured using the capacitance compensation circuitry.
Currents were corrected on line for linear leakage and capacitative
current by scaling the averaged response to four hyperpolarizing steps
of 5 mV amplitude obtained at a holding potential of 100 mV.
Blind patch-clamp recordings. In slices of adult rat dLGN,
voltage ramp recordings of INaP were performed
at 35 ± 1°C using an Axopatch 1D amplifier (Axon Instruments)
and pClamp. Series resistance in these experiments was 17 ± 1 M (n = 4).
Recording solutions. For Na+ current
characterization, the internal pipette solution contained (in
mM): CsF 120, HEPES 10, EGTA 10, MgCl2 2, CaCl2 1, Na2ATP 4, GTP 0.5, pH 7.3 with TEA-OH (Aldrich, Milwaukee, WI); osmolarity was adjusted to 290 mOsm. The
standard extracellular recording solution with physiological levels of
Na+ and the K+ and
Ca2+ channel blockers contained (in mM):
NaCl 120, sodium HEPES 16, KCl 2, glucose 10, TEA-Cl 20, CaCl2 1, 4-aminopyridine 2, MgCl2 4, NiCl2 0.5, CdCl2 0.1, pH 7.4; osmolarity was
adjusted to 300 mOsm. Steady-state and kinetic experiments on
INa were performed with a reduced extracellular
Na+ concentration (20 mM) to decrease
the amplitude of the current and so minimize the impact of possible
series resistance-induced errors. All experiments in neonate cats were
performed in 30 mM extracellular Na+.
The osmolarity of these solutions was maintained at 300 mOsm by
increasing the TEA-Cl concentration. In blind patch-clamp experiments in adult slices, NaHCO3 replaced sodium HEPES as the buffer
in the bathing solution.
Current-clamp recordings. For sharp electrode current-clamp
recordings, adult dLGN slices were placed in an interface-type chamber
and continuously perfused with a warmed (35 ± 1°C), oxygenated (95% O2, 5% CO2) medium
containing (in mM): NaCl 134, NaHCO3 16, KCl 2, KH2PO4 1.25, MgSO4 1, CaCl2 2, and glucose 10. Intracellular electrodes contained
1 M potassium acetate, recordings were performed using an
Axoclamp 2A, and current and voltage records were stored on a Biologic
DAT recorder (Intracel Ltd, Royston, UK). Some of these experiments
were performed in the presence of
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (ZD 7288) (kindly donated by Dr. P. Marshall, Zeneca, Macclesfield, UK).
Data analysis. Data were analyzed using the Clampfit program
of pClamp (Axon Instruments) and the mathematical transform and curve
fitting routines of Sigma Plot (Jandel Scientific, San Rafael, CA). For
the construction of steady-state activation and inactivation curves,
maximal conductances were estimated using the relation g = I/(Vs Vrev), where I is the measured
current, Vs the voltage step, and
Vrev the measured current reversal potential.
Estimated conductances were normalized and plotted against step
potential. Individual traces were fitted in Clampfit using the
Chebyshev algorithm to single exponentials of the form
y = ae-bt. Data points
were fitted with Boltzmann curves of the form y = 1/(1 + e(V1/2-V)/k) (where V1/2 is
the voltage of half-maximal activation, k is the steepness
constant), single exponentials of the form y = 1 aebt, and double exponentials of the
form y = 1 aebt + cedt. All potential values quoted in the
text and figures have been corrected for liquid junction potentials
(Barry and Lynch, 1991; Neher, 1992): 11 and 8 mV for low (20 mM) and high (130 mM) Na+
solution, respectively, calculated using Axoscope (Axon Instruments). All quantitative data in the text and figures are expressed as mean ± SEM unless stated otherwise, and statistical significance was tested using Student's t test.
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RESULTS |
The data presented in this paper are based on the following
preparations: dissociated neurons (n = 45) from neonate
rat, dissociated neurons (n = 7) from neonate cat,
neurons (n = 18) in slices of neonate rats, and neurons
(n = 14) in slices of adult rats.
Steady-state activation and inactivation
The kinetic and steady-state properties of
INa were determined using acutely dissociated
neurons from neonate rats, which enabled a better space clamp because
of the lack of an extensive dendritic arborization. In these
experiments the extracellular Na+ concentration was
also reduced to 20 mM to decrease the amplitude of the
current and further reduce voltage disparities introduced by series
resistance errors. To study the activation of
INa, neurons were clamped at a holding
potential of 111 mV, and depolarizing voltage steps of increasing
amplitude were delivered every 3 sec. At potentials more positive than
60 mV the voltage steps elicited rapidly activating inward currents
that inactivated within 10 msec (Fig.
1A1). The
elicited currents reached a peak at 23 ± 0.5 mV and reversed at
26.6 ± 4.2 mV (n = 6) (Fig.
1A2), a value close to the theoretically
determined reversal potential of 23 mV. This result, together with the
block by 1 µM tetrodotoxin (TTX) (data not shown),
defined this current as a TTX-sensitive neuronal
INa. The steady-state activation of
INa, constructed from six neurons, could
be fitted with a single Boltzmann curve (V1/2 = 29.84; k = 5.88) (Fig.
1A3). To compare
INa activation in different conditions/species
(see below) we also fitted the data of each neuron with a Boltzmann
curve. From this analysis we obtained a V1/2 of
29.8 ± 1.74 mV and a k = 5.48 ± 0.18 (n = 6) (Table 1).
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Figure 1.
Steady-state properties of
INa. A1, Currents
elicited with step depolarizations from a holding potential of 111 mV
to potentials of between 61 and 6 mV. A2,
Normalized current-voltage relation for INa
(n = 6). Currents from individual neurons were
normalized to their maximum amplitude and then averaged.
A3, Steady-state activation of
INa: normalized conductance values are
plotted against step potential. The points (n = 6)
are fitted with a single Boltzmann relation. B1,
Currents elicited by a step depolarization to 11 mV from holding
potentials of between 121 and 46 mV, to investigate INa inactivation (same neuron as in
A1). B2, These currents
were normalized to the current elicited from a holding potential of 121 mV and used to construct the steady-state inactivation. The plotted points (n = 6) are fitted with a single
Boltzmann function. The dotted line is the Boltzmann
curve fitted to INa activation (A3) to display the region of overlap between
the steady-state activation and inactivation curves
(INa window current). All experiments were
conducted in dissociated neonate rat neurons at room temperature.
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Inactivation was investigated using a protocol in which
INa was elicited with voltage steps to 11 mV
every 5 sec from different holding potentials.
INa was reduced when the holding potential was
more positive than 90 mV, and no current could be elicited from
holding potentials more positive than 40 mV (Fig.
1B1). The voltage dependence of
steady-state inactivation was plotted by normalizing
INa amplitude at different holding potentials to that elicited from a holding potential of 121 mV. The resulting points (from six neurons) could be fitted with a single Boltzmann curve
(V1/2 = 70; k = 5.88) (Fig.
1B2). Again, to compare
INa inactivation in different conditions/species
(see below) we also fitted the data of each neuron with a Boltzmann
curve and obtained a V1/2 = 70.04 ± 0.76 mV and a k = 5.8 ± 0.15 (n = 6)
(Table 1). Analysis of the steady-state activation and inactivation curves indicated the presence of a small area of overlap in a voltage
region centered around 50 mV, thus predicting the existence of a
"window component" of INa (Fig.
1B2).
Kinetics of fast activation and inactivation
Because of the fast activation of INa and
the uncertainty inherent in fitting such a rapid rising phase, the time
to peak was taken as an indicator of the rate of current activation.
Activation was voltage dependent (Fig.
2A,B1),
with the time to peak decreasing from 2.59 ± 0.79 msec at 41 mV
to 0.59 ± 0.01 msec at 9 mV (n = 4) (Fig.
2B1). INa decay
could be fitted with a single exponential function (Fig.
2A) and was found to be voltage dependent, with a 
that ranged from 2.19 ± 0.21 msec at 31 mV to 0.64 ± 0.07 msec at 9 mV (n = 4) (Fig.
2B2). The relationships of the time to
peak and of inactivation against membrane potential could not be
fitted with simple exponential functions. This finding is consistent
with classic models of channel activation (Hodgkin and Huxley, 1952),
so that our experimental data in the measured voltage range would be
located on the downstroke region of bell-shaped curves.
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Figure 2.
Fast activation and inactivation kinetics of
INa. A, Single exponential
decays of the form 1 eax well fitted the
fast inactivation of INa. Examples show fits of currents elicited by step depolarizations from 111 mV to 28.5, 26, 21, and 13.5 mV. B1, Plot shows the
time to current peak against step potential (n = 4). B2, Plot shows the of inactivation against step potential (n = 4; same cells as in
B1). Data are from dissociated neonate rat
neurons at room temperature.
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Onset of, and recovery from, inactivation
The onset of INa inactivation at a
particular potential will affect the action potential firing properties
of the neuron, because it defines the rate at which the channel
population enters an inactivated state. We investigated this process
using a two-voltage step protocol. Neurons were held at a negative
potential (108 mV), and INa was elicited by
10-msec-long depolarizing steps to 8 mV (Fig.
3A1). The onset of
inactivation at 68 mV was determined by stepping to this potential
for different durations before eliciting INa.
Times at the inactivating voltage were varied between 1 and 300 msec,
with currents normalized to the INa elicited
after 1 msec at 68 mV. Analysis of these data showed that the onset
of inactivation could be fitted with two exponentials with
1 = 37 msec and 2 = 76.9 msec (Fig.
3A2).
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Figure 3.
Onset of, and recovery from, steady-state
inactivation. A1, The onset of inactivation was
investigated using positive current steps to 68 mV from a holding
potential of 108 mV for varying lengths of time before eliciting
INa with a step depolarization to 8 mV.
A2, Currents were normalized to the amplitude of
INa elicited after a 1 msec delay at 68 mV
and plotted against time at the inactivating potential. Points
(n = 4) were fitted with two exponentials
(1 = 37 msec, 2 = 76.9 msec).
B, Recovery from inactivation. The time course of
recovery from inactivation at different holding potentials was
investigated using a two-pulse protocol. B1,
Plot of recovered current against lengths of delay of up to 30 msec
between depolarizing steps. Results are shown for three different
holding potentials: 111 mV (filled circles), 91 mV (open circles), and 71 mV
(filled triangles). All points represent data
from five neurons. Inset shows current traces at 71
mV. B2, Plot of recovery as in
B1 but for delay of up to 150 msec. Recovery at
111 mV and 91 mV was fitted with one exponential, whereas recovery
at 71 mV was fitted with the sum of two exponentials. Inset shows current traces at 71 mV over a 150 msec
period for the same cell as in B1. All
experiments were conducted in dissociated neonate rat neurons at room
temperature.
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The rate of recovery from inactivation also has fundamental
implications for the frequency and robustness of repetitive firing properties and was investigated using a two-pulse protocol.
INa was elicited with a 10 msec depolarizing
step to 11 mV, during which the current was inactivated. A second
test pulse was then delivered to elicit
INa, whereas the time between the two
pulses (
t) was varied between 1 and 150 msec to determine
the rate of recovery. The experiment was repeated at different holding
potentials to investigate the voltage dependence as well as the time
course of recovery (Fig. 3B). We observed that fast recovery
from inactivation, investigated by varying t in 1 msec
steps between 1 and 30 msec, was well fitted by single exponential
functions with = 5.52 msec and = 13.88 msec at holding
potentials of 111 mV and 91 mV, respectively. However, recovery at
71 mV was not well fitted with a single exponential (Fig.
3B1). Recordings with longer t durations showed that recovery was complete within 30 and 60 msec at
111 and 91 mV, respectively (Fig. 3B2). At
71 mV, however, recovery was not complete in 150 msec and was best
fitted with two exponentials (1 = 14.74 msec and
2 = 1.42 sec).
INa in TC neurons of the cat dLGN
Steady-state activation and inactivation of
INa in dissociated cat neurons was studied using
voltage protocols similar to those used in dissociated rat neurons
(Fig. 4A1,
A2). These data could be fitted with a single
Boltzmann curve for activation (V1/2 = 27.2;
k = 7.59) and inactivation (V1/2 = 65.7; k = 5.82), respectively. Indeed, the
parameters of the Boltzmann curves for INa
activation in five cat neurons show no difference in
V1/2 (27.82 ± 2.7 mV)
(p = 0.49) but a larger k (6.71 ± 0.25) (p < 0.005) compared with the rat,
whereas for the inactivation a slightly more depolarized
V1/2 (65.93 ± 1.0 mV)
(p < 0.05) but a similar k
(k = 5.92 ± 0.25) (p = 0.686) were observed (Fig. 4B) (Table 1). The kinetic
properties of INa in cat neurons were also
similar to those in rat neurons and included, for example, a time to
peak of 1.75 ± 0.29 msec and 0.76 ± 0.05 msec at 31 and 9 mV, respectively (Fig. 4C1), and a of
inactivation of 4.1 ± 1.56 msec and 1.67 ± 0.27 msec at
31 and 9 mV, respectively (n = 5, for all
measurements) (Fig. 4C2).
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Figure 4.
Steady-state and kinetic properties of
INa in dissociated neonate cat TC neurons at
room temperature. A1, Currents elicited from a
holding potential of 111 mV to test potentials of between 71 and
21 mV. A2, Currents elicited by a voltage step
to 11 mV from holding potentials of between 121 and 35 mV.
B, Steady-state activation and inactivation curves
obtained from measurements in five neonate cat neurons were fitted with
a single Boltzmann. Note the voltage dependence similar to the
corresponding curves for the neonate rat neurons (Fig.
1A3,B2) and the
presence of a window component. C1, Plot shows
the time to current peak against step potential (n = 5). C2, Plot shows the of inactivation
against step potential (n = 5; same neurons as in
C1).
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INaP in dLGN TC neurons
A persistent component (INaP)
of the Na+ current evoked by voltage step protocols
could be observed by increasing the gain of the amplifier, so that
INa was saturated. Both
INa and INaP were blocked
by TTX (1 µM) (n = 3) (Fig.
5A1). A
TTX-sensitive (n = 3), slow component of the
Na+ current (i.e.,
INaP) could also be seen when a voltage
ramp from 100 to 50 mV was delivered at a rate of 0.2 mV/msec (Fig.
5A2). Because the maximal amplitude of this
current was relatively small (~100 pA), this and the following
experiments were performed in extracellular solutions containing 130 mM Na+ to determine the amplitude and
properties of INaP under a physiological extracellular Na+ concentration.
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Figure 5.
TTX-sensitive INaP in
dLGN TC neurons. A1, Two long voltage steps to
63 and 38 mV (from a holding potential of 108 mV) elicit
sustained inward currents (INaP)
(solid traces). Dotted traces show that
after addition of 1 µM TTX to the perfusion medium, both
INa and INaP are
blocked. A2, A sustained Na+
current (INaP) could be elicited with
a ramp voltage protocol. The traces show the effect of 1 µM TTX on the inward current elicited by a voltage ramp
of 0.2 mV/msec from 118 mV. B1, The activation of INaP was investigated using long step
depolarizations. Traces show the current remaining up to
70 msec after step depolarizations to 88, 68, 53, 43, 33, and
23 mV. The saturating INa has been omitted
for clarity (the break in the record equals 40 msec). B2, Current-voltage relation for
INaP derived from voltage protocols as in
B1. The amplitude of the current was measured at
60 msec into the voltage step when the transient component had relaxed (n = 4). B3, Activation
curve for INaP, derived from the same cells as in B2. Points (n = 4) are fitted with a Boltzmann curve. All experiments were conducted in
dissociated neonate rat neurons at room temperature.
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The activation range of INaP was investigated by
applying long step depolarizations and measuring the amplitude of
INaP at 60 msec into the depolarizing step,
after INa relaxation (Fig. 5B1). Recordings were performed at a high gain
to enable reliable measurements of the sustained component.
INaP activation began at approximately 70 mV,
peaked at 39 ± 2 mV, and had an extrapolated reversal potential
of 4 ± 6 mV (n = 4) (Fig.
5B2). This reversal potential, however, is
probably an underestimation of the true reversal potential, because at
potentials more than 10 mV an outward current with properties similar
to those described by Alzheimer (1994) in pyramidal neurons of the
sensorimotor cortex was also activated. The activation of
INaP could be well fitted with a Boltzmann curve
(Fig. 5B3), characterized by a k of
9.09 and a V1/2 (56.93 mV) that was more
negative than that of INa (compare Fig.
1A3, B2). To
compare INaP activation in different conditions
(see below) we also fitted the data of each neuron with a Boltzmann
curve. From this analysis we obtained a V1/2 of
53.87 ± 3.05 mV and a k = 8.57 ± 1.89 (n = 4) (Table 1).
Properties and occurrence of INaP
Additional properties of INaP were
investigated in adult rat dLGN slices because of the greater amplitude
of INaP in this preparation (Fig.
6). The effect of the rate of voltage
change on the degree of INaP activation was
investigated by varying the rate of rise of voltage ramps, and the
amplitude of the pure INaP was then measured
after TTX and leak subtraction. We also studied the effect of ramp
rates in the physiological range from 0.1 to 0.5 mV/msec (Fig.
6A2). A rate of 0.1 mV/msec was
sufficient to elicit INaP, and the
amplitude of INaP evoked by a rate of 0.5 mV/msec was double the one that was evoked at the lowest rate. Note
that in this preparation, the fastest rates of rise were often
sufficient to activate INa as well (Fig.
6A1).
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Figure 6.
Steady-state properties of
INaP. A1,
TTX-subtracted inward currents elicited by voltage ramps of 0.5, 0.3, and 0.1 mV/msec from a holding potential of 100 mV in an adult rat
dLGN TC neuron. Note that ramps of higher rate cause transient spike as
well as activation of sustained current. A2,
Amplitude of elicited INaP is dependent on
the rate of voltage change. Plot of normalized INaP against ramp rise rate
(n = 4). B1, Amount of
INaP depends on the holding potential.
Traces show currents elicited during the ramp preceded
either by 3-sec-long voltage steps to 80, 60, and 50 mV, or by no
step. B2, Plot of normalized
INaP elicited with a ramp rate of 0.2 mV/msec against inter-ramp holding potential (n = 4). Experiments in A and B were conducted
in slices from adult rats at 35°C. C, Amplitude of
INaP in different species and preparations;
bar graph of INaP amplitude in dissociated
neonate rat TC neurons (n = 11), neonate
(n = 18) and adult (n = 4) rat TC neurons in slice, and dissociated neonate cat TC neurons
(n = 5). Currents were measured in 130 mM extracellular Na+ concentration for
all groups, apart from cat neurons, in which the extracellular
Na+ was 30 mM (see Results for
statistical significances). All data were obtained at room temperature
except those in adult rat slices (35°C).
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The possible inactivating effect of the holding potential or previous
cellular activity was also investigated. For this analysis, INaP was elicited with a voltage ramp from 100
to 50 mV while a conditioning potential was inserted between successive
ramps to determine its effect on the amount of
INaP evoked during the ramp (Fig.
6B1). The magnitude of
INaP was dependent on the conditioning potential
during the inter-ramp interval, with less current being evoked after
positive steps were increased. Thus, for example, only 60% of the
maximal INaP could be elicited after a
conditioning potential to 50 mV (Fig.
6B2).
To investigate possible subcellular, developmental, and species
differences, the peak amplitude of INaP elicited
during a 0.2 mV/msec voltage ramp from 100 to 50 mV was compared in
different preparations, all perfused with 130 mM
extracellular Na+ except for neonate cat (30 mM): neonate rat dissociated neurons, 52.27 ± 10.9 pA
(or 4.7 ± 0.5 pA/pF) (n = 11); neonate rat in slices, 86.22 ± 8.6 pA (n = 18); adult rat in
slices, 155.75 ± 34.7 pA (n = 4); and neonate cat
dissociated neurons, 92.8 ± 28.16 pA (or 4.6 ± 1.3 pA/pF)
(n = 5). The difference in INaP
amplitude between dissociated neonate rat neurons and neurons in
neonate rat slices was significant (p < 0.05),
indicating the presence of this current in TC neuron dendrites.
INaP amplitude in adult rat was significantly
greater than INaP in dissociated neonate rat
neurons (p < 0.001) and in neonate slice
(p < 0.01). The difference in
INaP between neonate rat and cat dissociated
neurons was not statistically significant, even when peak current
densities were compared (p = 0.89), but
note that in dissociated neonate rat neurons recorded under similar
conditions (i.e., in 30 mM extracellular Na+) INaP was almost
immeasurable (i.e., <5 pA).
Mechanism of INaP manifestation
To test the hypothesis that INaP could be
produced by a proportion of the Na+ channel
population that was noninactivating, we performed experiments (in
dissociated rat neurons and 20 mM
[Na+]o) in which 1 mg/ml papain
was included in the patch-recording pipette to cause removal of the
inactivation gate (Cota and Armstrong, 1992; Brown et al., 1994).
Because papain removed inactivation 15-20 min after commencement of
whole-cell recording, it was possible to obtain control data on
INa and then compare the current evoked after
removal of inactivation. In the low extracellular
Na+ solution used (20 mM),
INaP was practically immeasurable, but after 20 min of papain treatment a 0.2 mV/msec ramp protocol from 100 to 50 mV
elicited a large inward current (Ipapain)
that peaked between 40 and 30 mV (Fig.
7A), similar to the
ramp-evoked INaP in neurons that were recorded
with papain-free electrodes.
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Figure 7.
Removal of inactivation induces a current with the
properties of INaP. A, The
top traces display currents activated with a ramp
protocol immediately after breakthrough, and 20 min later, when an
internal solution containing 1 mg/ml papain was used. The bottom
trace displays the current induced by the action of papain and
was derived by subtracting the current recorded at t = 0 min from the current at t = 20 min. B, Papain causes the removal of fast
inactivation of INa in TC neurons.
Traces show currents elicited by step depolarizations
during control conditions soon after breakthrough, and 20 min after
recording with papain containing internal solution.
C1, Current-voltage relation of the
noninactivating current in papain-treated cells (filled
circles; n = 3) is superimposed on the
current-voltage relation of INaP taken from
Figure 5B2 (open circles) for
comparison. The dotted line at the base of the plot
shows the theoretical INa window current
calculated as described in the text. C2,
Activation curve for current in papain-treated cells
(filled circles; n = 3) is superimposed on the activation curve for
INaP taken from Figure 5B3 (open circles) for
comparison. All experiments were conducted in dissociated neonate rat
neurons at room temperature.
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Voltage step protocols were then used to obtain a more quantitative
analysis of the properties of Ipapain. We
observed that papain treatment transformed transient
INa currents elicited during step
depolarizations to noninactivating currents (Fig. 7B) that activated at more hyperpolarized potentials (around 70 mV) and peaked
at 40 mV (Fig. 7C1, C2). In
addition, there was no significant difference in the
V1/2 value between INaP
(filled circles in Fig. 7C2)
and Ipapain (56.92 ± 0.96 mV;
n = 3) (open circles in Fig. 7C2) (p = 0.86) or their
k value (Ipapain,
k = 6.31 ± 0.56 mV; n = 3)
(p = 0.28) (Table 1).
To test the hypothesis that INaP was a
manifestation of the window component of
INa, we compared
INaP with the theoretical INa window current. The measured
INaP (52.27 pA, see above) peaked at 39 mV,
and its amplitude was 2.5% of the peak INa
(2106 ± 208 pA; n = 9). The theoretical window
component was calculated from the product of the Boltzmann fits for the
steady-state activation and inactivation curves recorded with
papain-free electrodes (compare Fig. 1, A3 and
B2), and the mean conductance of
INa from neurons recorded in 130 mM
extracellular Na+. This predicted current
(dotted line in Fig. 7C1) peaked at
55 mV and had an amplitude of 4.7 pA. The difference in voltage
dependence and amplitude, therefore, suggests that
INaP is not a manifestation of the window
current of INa. We also calculated window
INa by using the results of a detailed
voltage-clamp analysis method (Tóth and Crunelli, 1995) in which
the steady-state activation curve is obtained without
"contamination" by activation or inactivation kinetics. Even in
this case, however, the amplitude of the calculated window
INa was <1 pA, and thus much smaller than the
measured INaP. Finally, evidence that
INaP was not a manifestation of window INa was obtained by the experiments in which
positive holding currents were seen to have an inactivating effect on
ramp-elicited INaP (Fig.
6B1), because window currents are not
expected to be affected by such potentials (Hirano et al., 1992).
Role of INaP in tonic and burst firing of
TC neurons
After the existence and properties of INaP
in TC neurons were established, we investigated the possible
physiological role of this current using current-clamp recordings from
adult rat dLGN slices (Fig. 8). The
membrane properties of these neurons were similar to
those described previously for TC neurons in identical recording
conditions (resting membrane potential: 62 ± 2 mV; apparent
steady-state input resistance: 191 ± 24 M; n = 10). The effect on tonic firing was investigated by recording from TC
neurons held at membrane potentials more than or equal to 60 mV, from
where low threshold Ca2+ potentials could not be
evoked by positive current steps (Fig. 8A1). Positive current steps were applied
in control conditions and in the presence of 1 µM TTX to
investigate the contribution of Na+ currents in this
region of the voltage-current relationship. TTX produced a block of
the action potentials and also a reduction in the extent of the
depolarization elicited by the positive current steps (Fig.
8A2). The greatest contribution of the
TTX-sensitive component (3.3 ± 0.5 mV; range, 2.5-4.0 mV;
n = 3) was at potentials closest to firing threshold
(Jahnsen and Llinás, 1984a,b).
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Figure 8.
Role of INaP in
tonic and burst firing of TC neurons. A1,
Voltage deflections produced by positive current steps from 60 mV in
control conditions and in the presence of 1 µM TTX.
A2, Steady-state voltage-current relation for
the neuron in A1 measured 20 msec before the end
of the current step (control: open circles; TTX:
filled circles). B1, Burst firing
produced by current steps of 40, 50, and 80 pA from a membrane
potential of 70 mV in control conditions (traces with action
potentials) and after the addition of 1 µM TTX. Note the
smaller and delayed low-threshold Ca2+ potential in
the presence of TTX. B2 and
B3 show responses in TTX (continuous
traces) at membrane potentials 68 mV and 72 mV,
respectively, on which the control responses recorded at 70 mV
(dashed traces) are superimposed for comparison. Note
that the reduction in amplitude and the delay of the low-threshold Ca2+ potential is evident even when the control
responses are compared with those evoked from 72 mV in which the
amplitude and the kinetics of the underlying
IT Ca2+ current are
larger and faster, respectively. This neuron was recorded in the
presence of ZD 7288 (200 µM). The amplitude of the action
potentials has been truncated for clarity, and the experiments were
conducted in slices from adult rats at 35°C.
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To investigate the possibility that INaP might
also have an involvement in the burst firing of TC neurons, neurons
were held at 70 mV, and positive current steps were then delivered to
activate the low-threshold Ca2+ potential and
associated burst-firing response. These experiments were performed in
the presence of ZD 7288 (200 µM), a specific blocker of
the hyperpolarization-activated inward current
Ih (Harris and Constanti, 1995; Williams et al.,
1997), to eliminate the effect of changes in this current on the
measured parameters (Pape, 1994; Hughes et al., 1996). For the neuron
shown in Figure 8B, a 40 pA current step in control
conditions elicited a robust low-threshold Ca2+
potential crowned by a burst of action potentials. After 1 µM TTX application, however, the same current step only
produced a much smaller and delayed low-threshold
Ca2+ potential (Fig.
8B1, left). With a current
step of 50 pA the low-threshold Ca2+ potential
recorded in the presence of TTX was also delayed compared with that in
control conditions (Fig. 8B1,
middle), but with a current step of 80 pA the low-threshold
Ca2+ potential profiles in control and TTX
conditions were almost indistinguishable (Fig.
8B1, right). The addition of
TTX, therefore, had two effects: (1) a large decrease (67 ± 6%;
n = 4) in the amplitude of the low-threshold
Ca2+ potential (measured at the lowest input current
required to evoke a full-blown Ca2+ potential in
control conditions) and (2) a significant increase in the latency of
the low-threshold Ca2+ potential, measured from the
onset of the current step (control: 83 ± 19 msec; TTX: 115 ± 18 msec; n = 4; p < 0.05; paired
t test). Because these changes in burst-firing properties
produced by TTX were observed within a narrow range of membrane
potentials (75 to 65 mV) and with relatively small positive current
steps (20-200 pA), extreme care was taken to ensure that cell
deterioration and shifts in membrane potential did not occur during the
course of these experiments. Results in control and experimental
conditions were carefully compared, and any neuron that displayed
deterioration in the quality of the recording was excluded from the
analysis. In addition, low-threshold Ca2+ potentials
were evoked in the presence of TTX not only from the same holding
potential as in the control conditions (i.e., 70 mV) (Fig.
8B1) but also at potentials slightly more
negative and positive (Fig. 8B2,
B3). Thus, positive current steps delivered from
68 mV (Fig. 88B2) elicited
low-threshold Ca2+ potentials that were markedly
reduced in amplitude and delayed compared with the control potentials
at 70 mV, suggesting that the effect observed at 70 mV in the
presence of TTX was not caused by a negative shift in membrane
potential during the experiment. Similarly, holding the neuron at 72
mV (Fig. 8B3), from where a larger
underlying IT Ca2+ current is
generated, still elicited low-threshold Ca2+
potentials that were delayed and displayed profiles that were not
superimposable on the control potentials.
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DISCUSSION |
The main conclusions of this study are that (1)
INaP exists in TC neurons; (2) a single
Na+ current type appears to underlie both
INa and INaP, with
the latter being formed by a noninactivating component of
INa; and (3) INaP contributes
to both tonic and burst firing of TC neurons.
Steady-state and kinetic properties of
INa
The properties of INa in TC neurons are in
general agreement with measurements of the action potential generating
INa in other CNS preparations. In particular,
for INa activation our values of
V1/2 and k fall within the range of
those reported by other investigators (from 39 to 28 mV and from
4.2 to 7.1, respectively), and similarly for the inactivation (from
90 to 60 mV and from 4.4 to 10.2, respectively) (Huguenard et al.,
1988; Sah et al., 1988; Fan et al., 1994; Magee and Johnston, 1995;
Safronov and Vogel, 1995). Single channel recordings have reported
INa decays to be best fitted with a single
exponential in motoneuron soma (Safronov and Vogel, 1995) or two
exponentials in hippocampal neuron dendrites (Magee and Johnston,
1995). In TC neurons INa decays could be well
fitted with a single exponential, which suggests therefore a single
type of Na+ channel population.
From the steady-state inactivation data it seems that at a typical
resting membrane potential of 60 mV only ~20% of the
Na+ channel population is available for activation
in TC neurons. A hyperpolarizing episode would therefore be expected to
remove inactivation and increase the available Na+
channel population, allowing for more robust action potential firing on
subsequent depolarization. This feature could explain why the frequency
of the burst firing evoked by low-threshold Ca2+
potentials in rat TC neurons of the dLGN and other thalamic sensory nuclei reaches a maximum of ~450 Hz, whereas in tonic firing the maximum frequency does not exceed 200 Hz (Huguenard and Prince, 1994;
Jahnsen and Llinás, 1984a; Williams et al., 1996). Indeed, it
would be interesting to investigate the onset and recovery from
inactivation of INa in intralaminar thalamic
nuclei in which burst firing frequencies of up to 1 kHz have been
observed (Steriade et al., 1993).
Properties of inactivation
From the experiments on the onset of inactivation at 68 mV, it
is evident that the amplitude of INa decreases
to ~50% of the initial value within 300 msec. This onset has a rapid
and a slow component. In neocortical neurons a slow entry into an inactivated state has been shown to have a marked effect on output firing patterns (Fleidervish et al., 1996), whereas the contribution of
this process to TC neuron firing remains to be determined. Recovery
from inactivation at holding potentials more negative than 70 mV
follow single exponential profiles, whereas at more positive (i.e.,
physiological) potentials an additional slower recovery from
inactivation is also seen. The inability to fit recoveries at these
potentials with a single exponential indicates that the relative values
of the of activation and the of inactivation converge. At more
negative potentials, single exponential fits show that activation is
much faster than inactivation (Tóth and Crunelli, 1996).
The decrease in firing frequency observed in TC neurons during long
periods of depolarization more than 40 mV (Williams et al., 1996;
Turner et al., 1997) will depend on the interplay between the onset of,
and the recovery from, inactivation as well as on the contribution of
K+ and high-threshold Ca2+
currents. The dynamics of this complex interplay in determining the
pattern of TC neuron output under different levels of excitability (Turner et al., 1997) could now be carefully examined using both experimental and simulation studies.
Mechanism of INaP manifestation
The three general hypotheses put forward to explain the occurrence
of INaP have been elegantly summarized in a
recent review (Crill, 1996). (1) INaP is a
manifestation of the window component of
INa; (2) INaP is a
distinct channel type that displays kinetic properties different from
INa; and (3) INaP
is produced by either a loss, or modulation, of inactivation of the
same channels that underlie INa.
The voltage dependence of INaP and window
INa in TC neurons was different, because the
maximal amplitude was observed at 40 mV and 55 mV, respectively.
Another difference was that the maximal amplitude of
INaP was ~2% of the whole-cell current,
whereas the calculated peak INa window current
would account for only 0.2% of the whole-cell current. In addition,
changes in the inter-ramp holding potential were seen to affect
markedly the amplitude of INaP elicited during a
subsequent voltage ramp, whereas window current amplitude should be
independent of the holding potential (Hirano et al., 1992). We
therefore conclude that INaP in TC neurons cannot be explained as a manifestation of the
INa window current.
Because of the lack of availability of toxins specific for different
subtypes of Na+ channels, it is only possible to
distinguish between channel types on the basis of kinetics or single
channel conductance. There is a clear difference in the activation
range of INaP and INa in
TC neurons that has been found in almost all other cell types studied
(French et al., 1990; Saint et al., 1992). This point, however, is
insufficient to prove the involvement of a different channel type and
could be attributed to the appearance of another "mode" of channel
gating (Alzheimer et al., 1993) or to a subpopulation of
Na+ channels having "lost" their ability to
inactivate. INaP could therefore arise from this
population of channels, as predicted from the Hodgkin and Huxley model
(Hodgkin and Huxley, 1952). The lack of inactivation has been suggested
as a likely mechanism in neocortical pyramidal neurons (Brown et al.,
1994).
The inactivation hypothesis was tested by including papain in the
recording pipette. This caused a removal of inactivation of
INa, transforming the rapidly
inactivating current into a sustained one. The greatly enlarged current
seen during ramp protocols with papain-containing electrodes had a
voltage dependence similar to the INaP evoked
during similar voltage protocols in control conditions.
Current-voltage relations of Ipapain
constructed with voltage step protocols were also similar to
INaP in untreated cells, displaying comparable
voltages of maximal amplitude and similar voltage dependence.
Activation curves constructed for Ipapain also
had V1/2 and k values that were not
different from those of INaP recorded with
papain-free electrodes. Cell-attached recordings in other neuronal
types have also failed to detect a distinct Na+
channel population underlying INaP (Alzheimer et
al., 1993; Magee and Johnston, 1995; Safronov and Vogel, 1995), and it
is clear that the only conclusive way of ruling out the involvement of a different Na+ channel subtype would be to perform
single channel recordings in TC neurons. In Purkinje neurons, a
"resurgent Na+ current" has recently been
described that is thought to derive from Na+
channels recovering from inactivation via an open state (Raman and
Bean, 1997). The properties of such a current, however, cannot explain
our findings that the greatest INaP contribution
occurs below firing threshold in current-clamp experiments and that
INaP is increased by the removal of inactivation
by papain. In view of the striking similarity of
Ipapain and INaP,
however, we suggest that the existence of a different channel type is
not required to explain INaP in TC neurons and
that at present the most parsimonious conclusion of our results is that
the appearance of INaP is caused by the absence
of inactivation in a subset of the Na+ channel
population underlying INa. The fact that one
channel type potentially underlies two very different
electrophysiological roles is in itself very interesting, as is the
possibility that the mechanism responsible for the loss of inactivation
may be under cellular control, a suggestion supported by our
experiments on the removal of inactivation by intracellular papain.
Physiological role of INaP
The TTX-sensitive INaP first activated at
membrane potentials around 70 mV, confirming the observation in other
systems that this current activates at potentials more negative than
those of INa. Indeed, the
V1/2 of INaP in TC
neurons is ~20 mV more negative than the V1/2
of the INa measured in this study and similar to
the one found in dorsal root ganglion cells and in hippocampal and
neocortical neurons (French et al., 1990; Brown et al., 1994; Baker and
Bostock, 1997). This relatively more negative activation of
INaP suggests a number of putative physiological
roles in TC neurons over a wide range of membrane potentials.
A maximum amplitude of ~150 pA in adult rat TC neurons suggests that
for a neuron with an apparent input resistance of 100 M, activation
of INaP could cause a depolarization of 10 mV. The current-voltage relation for INaP shows
that between 60 and 80% of this current will be activated in the
membrane potential range between 60 mV and action potential firing
threshold, predicting therefore that INaP would
have its greatest influence on membrane potential in this voltage
range. Indeed, the contribution of a TTX-sensitive,
INaP-mediated component to the voltage response of TC neurons to positive current steps was confirmed to be maximal in
the voltage range immediately below firing threshold, but its amplitude
was much less than predicted. This could be attributable to the large
effect of K+-dependent rectification in this voltage
region or to the reduction of the INaP elicited
during prolonged depolarization at potentials more than or equal to
60 mV (as seen in the conditioning potential experiments). Overall,
previous studies in guinea pig and bird TC neurons have shown a larger
amplitude (up to 10 mV) of a similar TTX-sensitive component, a result
that can be explained by the presence of a higher extracellular
K+ concentration and the consequent smaller
contribution of K+-dependent outward current in the
depolarizing responses observed in these studies (Jahnsen and
Llinás, 1984b; Ströhmann et al., 1994; Tennigkeit et al.,
1996). In contrast to the prominent role of INaP
in the high-frequency oscillations of cortical neurons, the presence of
this current is not essential for the expression of these oscillations
in TC neurons (Pedroarena and Llinás, 1997).
The activation of INaP at around 70 mV would
also suggest a possible physiological role at this voltage level. If
INaP activation was sufficiently rapid, then a
synergistic relationship of INaP and
IT in the generation of low-threshold
Ca2+ potentials and associated burst firing would be
expected. Despite the fact that INaP is
predicted to be small at potentials close to 70 mV, the effect of
removing this current on the amplitude and latency of a low-threshold
Ca2+ potential was remarkable. Indeed, although the
action of INaP on the low-threshold
Ca2+ potential was confined to a small range of
membrane potentials, it undoubtedly affected both the efficacy of burst
firing and the delay between triggering and firing. To the best of our
knowledge this is the first example of INaP
fulfilling an important amplification role in this voltage range, and
it suggests a potentially pivotal involvement of this current in the
burst-generating mechanism of TC neurons and a possible modulatory
target. Moreover, in view of the presence of dendritic T-type
Ca2+ channels in TC neurons (Zhou et al., 1997) and
their involvement in information processing (Guido and Weyland, 1995),
our findings of a contribution of INaP to burst
firing and its somatodendritic location suggest that this current plays
a major role in the integration of sensory and cortical inputs over a
relatively wide range of membrane potentials.
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Abstract
Introduction
