1Department of Neurology and Neurosurgery, Montreal Neurological Institute and McGill University, Montreal, Quebec H3A 2B4, Canada; 2Department of Experimental Neurophysiology, Istituto Nazionale Neurologico C. Besta, Milan 510, 20133 Italy; 3Department of Numerical Analysis and Computing Science, Royal Institute of Technology, S-100 44 Stockholm, Sweden; and 4Department of Psychology, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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Dickson, Clayton T.,
Jacopo Magistretti,
Mark H. Shalinsky,
Erik Fransén,
Michael E. Hasselmo, and
Angel Alonso.
Properties and Role of Ih in the
Pacing of Subthreshold Oscillations in Entorhinal Cortex Layer II
Neurons.
J. Neurophysiol. 83: 2562-2579, 2000.
Various subsets of brain neurons express a
hyperpolarization-activated inward current
(Ih) that has been shown to be instrumental in pacing oscillatory activity at both a single-cell and a network level. A characteristic feature of the stellate cells (SCs) of entorhinal cortex (EC) layer II, those neurons giving rise to the main
component of the perforant path input to the hippocampal formation, is
their ability to generate persistent, Na+-dependent
rhythmic subthreshold membrane potential oscillations, which are
thought to be instrumental in implementing theta rhythmicity in the
entorhinal-hippocampal network. The SCs also display a robust
time-dependent inward rectification in the hyperpolarizing direction
that may contribute to the generation of these oscillations. We
performed whole cell recordings of SCs in in vitro slices to investigate the specific biophysical and pharmacological properties of
the current underlying this inward rectification and to clarify its potential role in the genesis of the subthreshold oscillations. In
voltage-clamp conditions, hyperpolarizing voltage steps evoked a slow,
noninactivating inward current, which also deactivated slowly on
depolarization. This current was identified as
Ih because it was resistant to extracellular
Ba2+, sensitive to Cs+, completely and
selectively abolished by ZD7288, and carried by both Na+
and K+ ions. Ih in the SCs had
an activation threshold and reversal potential at approximately 45
and
20 mV, respectively. Its half-activation voltage was
77 mV.
Importantly, bath perfusion with ZD7288, but not Ba2+,
gradually and completely abolished the subthreshold oscillations, thus
directly implicating Ih in their generation.
Using experimentally derived biophysical parameters for
Ih and the low-threshold persistent Na+ current (INaP) present in
the SCs, a simplified model of these neurons was constructed and their
subthreshold electroresponsiveness simulated. This indicated that the
interplay between INaP and Ih can sustain persistent subthreshold
oscillations in SCs. INaP and
Ih operate in a "push-pull" fashion
where the delay in the activation/deactivation of
Ih gives rise to the oscillatory process.
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INTRODUCTION |
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The hyperpolarization-activated inward current
(Ih; usually referred to as
If in heart) has been implicated in
the pacemaking of both single-cell and network rhythmicity (for recent
reviews, see DiFrancesco 1993; Lüthi and
McCormick 1998
; Pape 1996
). Typically, this
current acts to promote depolarization after a hyperpolarizing event.
This, in combination with Ca2+ currents,
functions to induce low-threshold rhythmic discharge in a number of
neurons and thus contributes to brain rhythm generation (Llinás and Jahnsen 1982
; Lüthi et
al. 1998
; McCormick and Pape 1990
;
Steriade and Llinás 1988
; Steriade et al.
1993
). In contrast to Ca2+-dependent
oscillations, numerous studies have shown that some, mainly cortical,
neuronal populations can generate Na+-dependent
rhythmic subthreshold membrane potential oscillations that are thought
also to be implicated in the genesis of cortical rhythms (Alonso
and Llinas 1989
; reviewed by Connors and Amitai 1997
). The role of near-threshold conductances, including
Ih, in the generation of these
Na+-dependent subthreshold oscillatory events is
less clear.
A prominent case of Na+-dependent subthreshold
oscillatory activity is observed in the principal neurons from
entorhinal cortex (EC) layer II (Alonso and Klink 1993;
Alonso and Llinás 1989
). These glutamatergic
neurons, named by Cajal as the stellate cells (SCs) (Ramon y
Cajal 1902
), funnel most of the neocortical input to the
hippocampal formation via the perforant pathway (for review, see
Dolorfo and Amaral 1998
) and appear to be generators of
limbic theta rhythm (Alonso and García-Austt
1987a
,b
; Buzsáki 1996
; Dickson et
al. 1995
). In vitro current-clamp studies have shown that the
current-voltage relationship of EC layer II SCs is extremely nonlinear,
displaying robust inward rectification in both the depolarizing and
hyperpolarizing direction. Inward rectification in the depolarizing
direction is generated by a persistent subthreshold Na+ current
(INaP) (Magistretti and Alonso
1999
; Magistretti et al. 1999
) (for a recent
review on INaP, see also Crill
1996
) that has been shown to be necessary for the development
of the robust theta frequency subthreshold oscillations that the SCs
display (Alonso and Klink 1993
; Alonso and
Llinás 1989
). On the other hand, the time-dependent
inward rectification in the hyperpolarizing direction is affected by
extracellular Cs+ but not Ba2+ and
thus is likely to be generated by the nonspecific cationic current
Ih (Klink and Alonso
1993
). Given the properties and role of
Ih in pacemaking in other excitable
cells, it was proposed that this current also could contribute to the
genesis of subthreshold oscillations in SCs (Alonso and
Llinás 1989
; Klink and Alonso 1993
:
White et al. 1995
) although the exact nature of this
role was not specified.
Using the whole cell patch-clamp technique in the EC slice preparation,
the aim of the present study was to characterize the specific
properties of Ih in the SCs and to
examine the role of this current in the generation of subthreshold
membrane potential oscillations in these cells. In addition, a
simplified biophysical simulation based on the voltage- and
current-clamp data was used to study the interactions between
Ih and
INaP in the generation of such
oscillations. Our results indicate that the dynamic interplay between
the gating and kinetic properties of
Ih and
INaP is essential for the generation
of rhythmic subthreshold oscillations by the SCs. Given the key
position of the SCs in the temporal lobe memory system, modulation of
Ih in the SCs may have major
implications for the control of population dynamics in the entorhinal
network and in the memory processes it carries out. Some of these
results have been presented previously in abstract form (Dickson
and Alonso 1996, 1998
; Fransén et al.
1998
).
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METHODS |
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General
Brain slices were prepared from male Long-Evans rats (100-250
g, i.e., 30-60 days of age) as previously described (Alonso and
Klink 1993). Briefly, animals were decapitated quickly, and the
brain was removed rapidly from the cranium, blocked, and placed in a
cold (4°C) Ringer solution (pH 7.4 by saturation with 95% O2-5% CO2) containing (in
mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 CaCl2, 2 MgSO4, 26 NaHCO3, and 10 glucose. Horizontal slices of the
retrohippocampal region were cut at 350-400 µm on a vibratome (Pelco
Series 1000, Redding, CA) and were transferred to an incubation chamber
in which they were kept submerged for
1 h at room temperature (24°C). Slices were transferred, one at a time, to a recording chamber and were superfused with Ringer solution, also at room temperature. The chamber was located on the stage of an upright, fixed-stage microscope (Axioskop, Zeiss) equipped with a water immersion objective (×40-63: long-working distance), Nomarski optics,
and a near-infrared charge-coupled device (CCD) camera (Sony XC-75).
With this equipment, stellate and pyramidal-like cells could be
distinguished based on their shape, size, and position within layer II
of the medial entorhinal cortex (Fig.
1A) (Klink and Alonso
1997
). Stellate cells (SCs) were selected for whole cell
recording.
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Recording
Patch pipettes (4-7 M) were filled with (in mM)
140-130 gluconic acid (potassium salt: K-gluconate), 5 NaCl, 2 MgCl2, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic
acid (HEPES), 0.5 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 ATP (ATP Tris salt), and 0.4 GTP (GTP Tris salt), pH
7.25 with KOH. In additional experiments performed to assess the
contribution of chloride ions to Ih, a
modified intracellular solution was made containing (in mM) 120 K-gluconate, 10 KCl, 5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATP-Tris,
and 0.4 GTP-Tris, pH 7.25 with KOH. The liquid junction potential was
estimated following the technique of Neher (1992)
. In
brief, the offset was zeroed while recording the potential across the
patch pipette and a commercial salt-bridge ground electrode (MERE 2, WPI, Sarasota, FL) when the chamber was filled with the same
intracellular solution as used in the pipette. After zeroing, the
chamber solution was replaced with the extracellular recording
solution, and the potential recorded was used as an estimate of the
liquid junction potential. Using this method, we recorded a value
between 2 and 3 mV. Membrane potential values reported herein do not
contain this correction.
Tight seals (>1 G) were formed on cell bodies of selected EC layer
II SCs, and whole cell recordings were made by rupturing the cell
membrane with negative pressure. Both current- and voltage-clamp recordings were made with an Axopatch 1D patch-clamp amplifier (Axon
Instruments, Foster City, CA). For current-clamp recordings, the
low-pass filter (
3dB) was set at 10 kHz, whereas for voltage clamp,
it was set at 2 kHz. All current- and some voltage-clamp experiments
were stored by PCL coding on VHS tape (Neurocorder, Neurodata, New
York), and all voltage-clamp experiments were stored on computer by
digital sampling at 4 kHz, using pClamp software (V6.0, Axon
Instruments). Data stored on VHS tape was digitized and plotted
off-line by sampling at 20 kHz using Axoscope software (V1.1, Axon Instruments).
The identification of neurons as SCs was confirmed by current-clamp
recordings demonstrating the presence of robust inward rectification
with hyperpolarizing current pulses in addition to the presence of
subthreshold membrane potential oscillations at depolarized levels
(Fig. 1, B and C) (Alonso and Klink
1993). SCs fulfilling the following criteria were considered
acceptable for further analysis: stable membrane potential less than
50 mV, input resistance >75 M
, overshooting spike, and a balanced series resistance <20 M
compensated between 60 and 80%.
Solutions
Various salts and drugs were added directly to the
perfusate from concentrated stock solutions during experiments.
Divalent cations such as Ba2+,
Co2+, or Cd2+ were added to
a modified Ringer solution without phosphates or sulfates. To isolate
Ih to compute activation curves using
the tail current method (see RESULTS), the following
solution was used (in mM): 80 NaCl, 40 tetraethylammonium chloride
(TEA-Cl), 5 KCl, 4 4-aminopyridine (4-AP), 2 MgCl2, 2 BaCl2, 2 CoCl2, 1 CaCl2, 0.2 CdCl2, 26 NaH2CO3, and 10 glucose and
1 µM tetrodotoxin (TTX). Lowering the extracellular concentration of
sodium ions (NaCl) was achieved using equimolar
N-methyl-D-glucamine substitution in a no
phosphate/sulfate Ringer solution. Alterations in the concentration of
potassium ions (KCl) was achieved in the same way in a no
phosphate/sulfate Ringer solution with a consistent concentration (119 mM) of NaCl. To prevent the influence of synaptic transmission on the
subthreshold membrane behavior in current-clamp recordings,
6-cyano-7-nitoquinoxaline-2,3-dione (CNQX: 10 µM), DL2-amino-5-phosphonopentanoic acid (AP-5: 50 µM),
bicuculline methiodide (BMI: 10 µM), and 2-hydroxysaclofen (2-OH
saclofen:100 µM) were added to the Ringer solution. To block
Ih, CsCl (1-6 mM) or ZD7288 (100 µM) was added directly to the Ringer solution. All salts were
purchased from BDH (Toronto, CA), whereas TEA-Cl, 4-AP, TTX, and BMI
were purchased from Sigma (St. Louis, MO). CNQX, AP-5, 2-OH-saclofen,
and ZD7288 were purchased from Tocris Cookson (UK).
Analysis
Traces were plotted and measurements made with the use of pClamp (Clampfit) and Origin (Microcal, Northampton, MA) software packages. Curve fitting of subtracted traces was conducted with pClamp software. The fittings were made from a time point 15 ms after the application of the hyperpolarizing voltage step so as to minimize any capacitive or membrane charging transients. All curve-fitting procedures were optimized using the least sum of squares method. The standard deviation between the fit and the data were used to estimate the goodness of the fit. Autocorrelational analysis was conducted with Matlab (Mathworks, Natick, MA). Spectral (Fourier) analysis was conducted using both Origin and Matlab.
Biophysical simulation
A simple Hodgkin-Huxley model was assumed for describing
Ih activation and deactivation. We applied
the basic relationship
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(1) |
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(2) |
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(3) |
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(4) |
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(5) |
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The basic equations used for describing
INaP were the same as used for
Ih (see preceding text, Eqs.
1 and 2).
GNaP(V) was modeled
according to the voltage-dependence data reported by Magistretti and Alonso (1999). INaP
activation was assumed to be instantaneous.
Kinetic and voltage-dependence parameters concerning
Ih and
INaP were used in a simplified model
of an EC SC aimed at reproducing the subthreshold oscillatory behavior
of membrane potential in these same neurons. In this model, the neuron
was considered as monocompartmental, and its membrane conductance
consisted of Gh, GNaP, and a linear leakage conductance
(Gl) whose current reversed at the
equilibrium potential for K+. The parameters of
the equations describing conductance kinetics and voltage dependence
were given the same numerical values as returned by the analysis of the
relevant experimental data (see RESULTS).
Na+ and K+ reversal
potentials had the theoretical (Nernst) values calculated for the ionic
conditions employed in our current-clamp experiments (VNa = +87 mV,
VK = 83 mV). The reversal potential
for Ih
(Vh =
20 mV) and the amplitude ratio
between the fast and slow kinetic components of
Gh
(Gh1Max /Gh2Max
= 1.85) also matched exactly the experimentally observed average
values. Only the absolute values of maximal conductances
(GMax) were adjusted until a good concordance between simulations and experimental observations was
achieved. In the simulations here illustrated,
GhMax,
GNaPMax, and
GlMax equaled 98.0, 17.4, and 78.0 pS/pF, respectively. These values compared reasonably, namely within a
factor of 2, with the experimentally measured values.
Numeric solution of the differential equations was achieved by
the use of a one-step Euler integration method. The integration step
size was 0.25 ms. Preliminary tests on the adequacy of this integration
method were carried out by reducing the step size by 25 times, which
revealed an optimal convergence. The simulation programs were compiled
using QuickBASIC 4.5 (Microsoft). Data were analyzed using Origin.
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RESULTS |
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The results presented in this study were based on a database of
131 EC layer II SCs intracellularly recorded under whole cell patch
conditions and met the criteria specified in METHODS. The studied neurons were identified as SCs by their gross morphological characteristics (Klink and Alonso 1993) as afforded by
direct visualization of their somata and proximal dendrites (Fig.
1A) but mainly by their characteristic electrophysiological
properties (Alonso and Klink 1993
; Alonso and
Llinás 1989
) (Fig. 1, B-D). Indeed, as
illustrated in Fig. 1, patched SCs demonstrated qualitatively the same
electroresponsive properties that distinguish SCs recorded with sharp
electrodes. First, the patched SCs demonstrated robust time-dependent
inward rectification in the hyperpolarizing direction. As shown in Fig.
1B, the membrane voltage responses to hyperpolarizing current pulses did not monotonically reach a steady value but displayed, after a certain delay, large amplitude "sags" back to
more depolarized values. Second, the action potential of the patched
SCs also demonstrated the characteristic fast after hyperpolarization (arrowhead Fig. 1C) followed by a depolarizing
afterpotential and a medium after hyperpolarization. Finally, and most
importantly, patched SCs also developed rhythmic subthreshold membrane
potential oscillations and demonstrated cluster discharge when
depolarized with DC current in the membrane potential range between
55 and
50 mV (Fig. 1D, 1-3). At an average
membrane potential of
52 ± 1 mV, the peak frequency of these
membrane potential oscillations as determined by Fourier analysis
averaged 3.1 ± 0.7 Hz (n = 12). The SCs had an
average resting membrane potential of
55 ± 3 mV and an input
resistance of 113 ± 40 M
.
Although not further treated, in some instances, neurons other than SCs
were recorded from. Pyramidal-like cells of EC layer II
(n = 10), could be distinguished from SCs based on
their pyramidal shape and their limited expression of time-dependent
inward rectification (Klink and Alonso 1993). Layer III
pyramidal cells (n = 3) were distinguishable based on
their qualitatively smaller size, their high-input resistance (217 ± 67 M
) and the absence of time-dependent inward rectification
(Dickson et al. 1997
).
Hyperpolarization-activated, time-dependent inward rectification in SCs corresponded to a slow, noninactivating inward current
As illustrated for a typical SC in Fig.
2, the depolarizing sags that developed
on membrane hyperpolarization in current-clamp conditions
(A, ) were paralleled by the development of a slow inward
current on step hyperpolarization under voltage-clamp conditions (B,
). Note that the time course and amplitude of this
inward relaxation was overtly voltage dependent (see following text). In all cases, analysis of the subthreshold input-output relations under
current-clamp revealed that the steady-state voltage-current (V-I) curve (Fig. 2C;
) showed a marked upward
bending over the entire voltage range (
60 to
120). Similarly,
analysis of input-output relations under voltage-clamp revealed that
the steady-state current-voltage curve (ssI-V; Fig.
2D,
) showed a robust inward shift, as compared with the
instantaneous current-voltage curve (Fig. 2D,
), that grew steadily with membrane hyperpolarization. The slow inward current
relaxations were associated with a membrane-conductance increase
because the instantaneous current flowing at the break of the
hyperpolarizing commands was larger than that recorded on first jumping
to the command potential (see Fig. 4A). Thus SCs do possess
a robust time-dependent hyperpolarization activated conductance
(Gh).
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Pharmacological block of inward rectification
In addition to a time-dependent inward rectifier such as
Ih, many neurons also possess a fast
inward rectifier K+ current
(IKir) (reviewed by Hille
1992). It has been shown that in many cells bath application of
Ba2+ and Cs+ can be used to
pharmacologically dissect Ih from
IKir because Ba2+ blocks IKir
and not Ih, whereas
Cs+ blocks both
IKir and
Ih (Hagiwara et al. 1976
,
1978
). In agreement with this, in all SCs tested
(n = 8), bath application of Ba2+
(0.5-2 mM) had no effect on Ih (Fig.
3, A-C), although it did block the small inward bending of the instantaneous I-V
relationship that was always observed at potentials negative to about
80 mV in control conditions (Fig. 3C, squares). This
Ba2+ effect suggests the presence of a minor
IKir in the SCs. In contrast to
Ba2+, in all SCs tested (n = 10),
bath application of Cs+ (1-6 mM) always produced
a substantial decrease in Ih (though never a complete Ih block). This
decrease was assessed by expressing the percentage decrease in the
difference between the instantaneous and steady-state current at
potentials between
60 and
80 mV before and after application of
Cs+ (cf. Ishii et al. 1999
). It
was dose dependent and ranged from 60 to 75% for a concentration of 2 mM Cs+ (n = 5) that produced
close to maximal effects.
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Given that Cs+ produced only a partial block of
Ih in the SCs, we assessed the effects
of the novel bradycardic agent ZD7288, which has been reported to be a
potent blocker of Ih in other cells
(BoSmith et al. 1993; Harris and Constanti
1995
; Maccaferri and McBain 1996
;
Williams et al. 1997
). As illustrated in Fig. 3
(D and E), in all cases tested
(n = 9), bath application of ZD7288 (>10 min; 100 µM) always resulted in a complete and irreversible block of
Ih. When the cells were held at
60
mV (about resting level), application of ZD7288 always resulted in an
outward shift of the holding current (mean =138 ± 52 pA,
n = 5), indicating that
Gh is active at the resting membrane
potential (see following text). Significantly, ZD7288 did not abolish
the small inward shift of the instantaneous I-V relation
below
80 mV (Fig. 3G) and thus whereas ZD7288 fully
blocked Ih, it did not affect
IKir. Although small, the remaining
fast inward rectification, however, could be blocked fully by the
further addition of Ba2+ that also caused a
decrease in slope conductance due to its blocking action on leak
currents (n = 3; Fig. 3, F and
H).
Activation of Ih
We estimated the activation curve of the membrane
conductance underlying Ih
(Gh) by applying two different
protocols (Fig. 4). In the first
protocol, a modified Ringer solution (as specified in
METHODS) was used. The activation curve of
Gh was estimated from the peak
amplitude of the tail currents recorded at about 40 mV
(n = 8) or at about
60 mV (n = 5)
after a series of hyperpolarizing voltage-clamp steps from a holding
potential in the range of
45 to
30 mV (Fig. 4,
A-C). When stepping back to
60 mV, the zero current level was the tail current amplitude after the most depolarized voltage step (at least
40 mV). Tail current amplitudes were
normalized to the maximal value (Imax)
and plotted as a function of the membrane potential during the
hyperpolarizing prepulse. In all cases (n = 13), the
data were well fitted with a Boltzmann equation of the form
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For comparison, in four cells we applied slow (<10 mV/s) 100 mV
hyperpolarizing voltage ramps from a holding potential of 30 mV in
control and after block of Ih with
ZD7288 (Fig. 4D). In these experiments, 1 µM TTX, 2 mM
Co2+, and 2 mM Ba2+ were
added to the control Ringer. Subtraction of the ZD7288 I-V curve from the control curve yielded the steady-state
Ih I-V relationship from
which we estimated Gh according to the
formula Gh=
Ih/(Vm
Vh) where
Vh is the reversal potential for
Ih estimated to be
21 mV (see
following text, Fig. 6). The resulting values were normalized to the
maximal conductance (Gmax; 10.4 ± 2.3 nS) and plotted against Vm. In
all cases the curves were well fitted with a Boltzmann equation as
in the preceding text. This ramp analysis yielded a
V1/2 of
76 ± 4 mV and a slope
factor of 12.1 ± 2, which were not significantly different to
those obtained by tail current analysis by two-tailed
t-tests [t(15) = 0.36, P > 0.05; t(15) =
0.85, P > 0.05].
Time course of activation and deactivation
As stated previously, the rate of activation of
Ih increased sharply with
hyperpolarization (e.g., Fig. 2B). This qualitative observation was further explored in a more quantitative manner. To
maximize the accuracy of our kinetic analysis, we isolated Ih by subtracting from control current
traces evoked by hyperpolarizing voltage-clamp steps, the current
traces evoked to the same potentials in the presence of the selective
Ih blocker ZD7288 (n = 5; Fig. 5A). Over the whole
voltage range tested, the Ih current
relaxations were best fitted with a double exponential function of the
form
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An equivalent method as the one described in the preceding text was
conducted to study the rate of deactivation of
Ih isolated with the use of ZD7288
(Fig. 5B). Isolated Ih
current traces evoked by depolarizing voltage steps from a holding
potential of 60 mV were also well fitted by a double-exponential
function. As for the time constants of activation, both the first and
second time constants of deactivation were found to be voltage
dependent, becoming faster with increasing depolarization (Fig. 5,
C and D). The first time constant of deactivation
ranged from 23 ± 9 to 58 ± 13 ms for voltage steps to
40
and
50 mV, respectively. The second time constant of deactivation
ranged between 241 ± 38 and 326 ± 58 ms for the same
voltage steps. The amplitude of the fast time constant was roughly 1.25 that of the slower, and this ratio remained constant over the voltage
range tested.
Reversal of Ih
Estimation of the reversal potential of
Ih was achieved by two different
methods, which took advantage of the fact that at 80 mV,
Gh was strongly activated and did not
show time-dependent inactivation (Fig. 6,
A and B). In all
experiments, the superfusing Ringer solution contained 1 µM TTX, 2 mM
CoCl2, and 2 mM BaCl2. Thus
in the first method, we estimated the reversal potential of
Ih
(Vh) from the intersection of the
instantaneous (chord) current-voltage relationships recorded at holding
potentials of
80 and
40 mV (i.e., in the presence and absence of
Ih; Fig. 6C) (Mayer
and Westbrook 1983
). In 17 neurons examined, this method provided an average value for Vh of
21 ± 5mV.
|
To support the preceding estimation, we used a second method in which
we took advantage of the fact that Ih
is selectively and fully blocked by ZD7288 (see preceding text). Chord
conductance measurements were made from voltage steps from a holding
potential of 80 mV before and after block of
Ih using ZD7288 and the instantaneous I/V relationships in both conditions were constructed (Fig. 6, D-F). In eight neurons examined, the average voltage at
which the linear fits for both plots intersected, i.e., the reversal potential for Ih, was
22 ± 6 mV, a value that was not significantly different from that found with
the tail current analysis method above [t(23) = 0.44, P > 0.05].
Ionic basis of Ih
The fact that in the SCs Ih
reverses at about 20 mV suggests that, as in other neurons
(Crepel and Penit-Soria 1986
; Halliwell and Adams
1982
; Mayer and Westbrook 1983
; McCormick
and Pape 1990
; Spain et al. 1987
;
Takahashi 1990
), this hyperpolarization-activated inward
current might be carried by a mixture of both Na+
and K+ ions. Indeed, increasing the extracellular
concentration of K+
([K+]o) (Fig.
7) produced an increase in
Ih (with no change in the Gh activation curve; not shown) as
well as an increase in instantaneous conductance. As expected for
K+ being an important carrier for
Ih, an increase in
[K+]o from 1 to 10 mM
produced an average positive shift in
Vh of 10 ± 4mV
(n = 4, Fig. 7D).
|
On the other hand, reductions in the concentration of extracellular Na+ from control levels (151 mM) to 26 mM reversibly reduced the amplitude of Ih (Fig. 8) without changing the activation properties of the conductance underlying this current (not shown). Concomitant with this reduction, Vh shifted in the hyperpolarizing direction by an average of 21 ± 5mV (n = 5). These results indicate that Na+ ions also largely contribute to Ih.
|
Finally, a number of neurons (5) were recorded using a modified
intracellular solution containing an additional 10 mM
Cl in the pipette solution (see
METHODS). Although in these cases, the chloride reversal
potential was theoretically shifted by ~20 mV in a positive
direction, no significant difference was observed in either the average
reversal potential [
23 ± 6 mV; t(20) =
1.48,
P > 0.05] or the activation properties of
Ih (not shown). Thus using the
Goldman-Hodgkin-Katz equation and an estimated Vh of
21.5 mV, we calculated a
permeability (conductance) ratio for Na+ and
K+
(pNa+/pK+) of ~0.4 for
Ih in the SCs.
Role of Ih in membrane potential oscillations
Given the overlap between the activation range of
Gh (threshold at about 45 mV), and
the voltage range at which subthreshold membrane potential oscillations
occur in SCs (
60 to
50 mV), we sought to define the involvement of
Ih in these oscillations by exploring
the effects on them of Cs+, ZD7288 and
Ba2+. Because these agents, particularly
Cs+ and Ba2+, greatly
enhance spontaneous synaptic events, we carried out this analysis
during synaptic transmission block with CNQX (10 µM), AP5 (50 µM),
bicuculline (10 µM), and 2-OH-saclofen (100 µM). In line with a
role of Ih in the generation of the
rhythmic subthreshold oscillations, we observed that the addition of
Cs+ (1-2 mM; n = 4) to the
superfusate resulted in a progressive disruption (and slow-down) of the
oscillations. However, as previously reported (Klink and Alonso
1993
), some trains of subthreshold oscillatory activity could
consistently be observed in the presence of Cs+.
This result might be interpreted as suggestive that, in addition to
Ih, another conductance operating in
the subthreshold range, such as the M current, may play a major role in
the generation of the rhythmic subthreshold oscillations by the SCs.
Alternatively, it also might be that the expression of subthreshold
oscillatory activity by the SCs is rather insensitive to the level of
Ih expression and that a major
decrease in Ih is necessary to abolish
the oscillations. To explore these possibilities, we first tested the
effects of the more potent Ih blocker
ZD7288. In all cells, application of 50-100 µM ZD7288 always
resulted in membrane hyperpolarization (9 ± 4 mV;
n = 8) concomitant with the block of the typical
depolarizing voltage sag evoked by hyperpolarizing current pulses.
Similarly to Cs+, ZD7288 always produced a
progressive disruption of the oscillations, though, in contrast to what
was observed with Cs+, this disruption always
proceeded to a complete block (Fig. 9, A-C). Although these data suggest that, indeed, a major
block of Ih is necessary to completely
abolish the oscillations, it could be argued that the blocking effect
of ZD7288 might have been due to a nonselective action of the drug on
another conductance operating in the oscillatory range. To exclude this
possibility, we performed a series of voltage-clamp experiments in
which we examined the effects of ZD7288 on the outward current
relaxations evoked by a series of depolarizing voltage-clamp steps from
60 mV (about resting level) to the voltage range where subthreshold oscillations develop (
55 to
50 mV; Fig.
10A) and up to the
Gh activation threshold (
45 mV).
These experiments were conducted in the presence of 1 µM TTX and 2 mM
Co2+. As shown in Fig. 10, B-D,
ZD7288 (100 µM) always caused a robust outward shift in the holding
current and a complete and selective block of both the outward current
relaxations in response to membrane depolarization as well as the
associated tail currents on return to the holding potential
(n = 4). In contrast, there was a nearly perfect
overlap between the traces at
45 mV, the threshold for activation of
Ih, before and after ZD7288. This indicates
that, in the voltage range from
60 to
45 (which includes the
voltage range at which the membrane potential oscillations occur) the action of ZD7288 was specific for Ih.
Thus the block of the oscillations by ZD7288 cannot be attributed to a
nonspecific effect of the drug.
|
|
Finally, it also might be argued that the disappearance of sustained
subthreshold oscillations with ZD7288 resulted from the membrane
conductance decrease due to the Ih
block and not by the Ih block per se.
This possibility was tested with the use of Ba2+
(1-2 mM; n = 7), which, in contrast to ZD7288, does
not affect Ih (cf. Fig. 3) but which,
similarly to ZD7288, also produces a major decrease in membrane
conductance. Importantly, and in sharp contrast to the ZD7288 results,
bath superfusion with Ba2+ resulted in both a
significant increase in the amplitude [1.2 ± 0.5 mV;
t(4) = 2.7; P < 0.05] and a
significant decrease in the frequency [1.3 ± 0.1 Hz;
t(6) = 8.8; P < 0.01] of the
subthreshold oscillations (Fig. 9, D-F). In consequence,
the above indicates that Ih plays an
essential role in the generation of rhythmic subthreshold oscillations
by the SCs and that leak conductances can modulate their amplitude and
frequency through their effects on passive membrane properties.
Role of Ih and INaP in the generation of subthreshold oscillations
Although the preceding experimental data indicate that
Ih is necessary for the genesis of
subthreshold oscillations by the SCs, previous studies have shown that
these oscillations are also dependent on the activation of a
subthreshold persistent Na+ current
(INaP) (Alonso and Llinás
1989). To generate an oscillatory phenomenon, a process is
needed the action of which feeds-back to slow down the rate of the
process itself and, most critically, a delay in the execution of the
feedback. In SCs, the slow kinetics of activation and deactivation of
Ih potentially can implement such a
feedback process. To further clarify the role of
Ih in the generation of subthreshold
oscillations by the SCs and to complement the preceding experimental
data, we next implemented a simplified biophysical simulation of the
subthreshold membrane voltage behavior of these neurons. Using
classical Hodgkin-Huxley formalism, a theoretical reconstruction of the
biophysical properties of Ih first was
carried out. To be consistent with our experimental data indicating
both a fast and a slow kinetic component of
Ih (see preceding text; Fig. 5), we
constructed activation plots of the corresponding fast and slow
conductance components (Gh1 and
Gh2, respectively; Fig.
11A, 1 and
2) from which the m
i curves were derived directly by using a standard Boltzmann fitting (see
METHODS and legend of Fig. 11). The voltage dependence of the fast and slow rate constants (
i and
i; Fig. 11C, 1 and
2) was estimated from the corresponding time constants of
activation and deactivation (
i; Fig.
11B, 1 and 2) and the
m
i curves, as explained in detail
in METHODS.
|
The derived parameters for the kinetics and voltage dependence of
Ih and those for
INaP as described previously
(Magistretti and Alonso 1999) then were incorporated in
a single compartment model of the SC (see METHODS). The
model then was explored to test whether it could reproduce
characteristic current-clamp phenomena such as the sag in membrane
potential during hyperpolarizing current steps and the generation of
subthreshold membrane potential oscillations.
Voltage responses to hyperpolarizing current steps in the model SC are
illustrated in Fig. 12A.
Note that the model cell did display the typical delayed
large-amplitude depolarizing sags in response to membrane
hyperpolarization as well as robust rebound potentials at the break of
the hyperpolarizing current pulses. More importantly, as shown in Fig.
12B, the model SC also developed sustained rhythmic membrane
potential oscillations in response to DC membrane depolarization from
its resting level to about 53 mV. The combined experimental and model
work thus demonstrates that in the SCs the interplay between
Ih and
INaP is essential for the generation
of sustained rhythmic subthreshold membrane potential oscillations.
|
As illustrated in Fig. 12C, the use of the model SC also allowed us to understand the dynamics of the interplay between INaP and Ih (as well as GNaP and Gh) during the oscillatory cycle. Note that at the trough of an oscillation (1st vertical dashed line) INaP and GNaP are at a minimum, whereas Ih and Gh are approaching, but not yet at, their respective maxima. This occurs after a certain delay (Gh maximum lags GNaP and Vm minimum by 21 ms at 3 Hz), and thus the attainment of a maximum level by Ih coincides with the initiation of a depolarizing phase. As depolarization proceeds, INaP rapidly increases because GNaP becomes progressively activated. In turn, the depolarization boosted by INaP leads to Gh deactivation and thus a decrease in Ih, which slows down and eventually stops the depolarization. Note that at the peak of the oscillation (2nd dashed line) Ih and Gh (similarly to what reciprocally occurs at the trough) are approaching, but they are still not at, a minimum. This occurs after the peak. Thus the deactivation of Gh is now responsible for initiating the repolarizing phase of the oscillation. Membrane hyperpolarization leads to a rapid decrease in INaP that boosts further hyperpolarization. Eventually membrane hyperpolarization leads to the new activation of Ih and a new oscillatory cycle is initiated.
It can be observed from the traces in Fig. 12C that INaP essentially changes instantaneously with changes in Vm, whereas changes in Ih follow with a certain delay (caused by its time-dependent properties). This differential behavior may be better understood by plotting INaP and Ih as a function of Vm as illustrated in Fig. 13. Note that although the Ih curve demonstrates substantial hysteresis, the INaP curve demonstrates very little to none. The increase and decrease in INaP during the depolarizing and hyperpolarizing phase of an oscillation maintains an almost perfect linear relationship with changes in Vm. In contrast, the trajectory followed by Ih during the depolarizing phase of the oscillation is substantially different from that followed during the hyperpolarizing phase. Ih decreases slowly during the initial part of the depolarizing phase but this rate of decrease accelerates as the membrane potential approaches its peak value. Similarly, during the hyperpolarizing phase of the oscillation Ih increases slowly during the initial portion, and this rate of increase accelerates as the membrane potential approaches its minimal value. Basically, the hysteresis introduced by the kinetic properties of Ih implement a delayed feedback mechanism to the voltage changes led by INaP that allows sustained oscillatory activity to occur.
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DISCUSSION |
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The present results demonstrate that the robust
hyperpolarization-activated time-dependent inward rectification
displayed by the stellate cells from EC layer II is due to an inward
current that we identified as Ih on
the basis of its pharmacological and biophysical profile as described
in other excitable cells (see recent revisions by Clapham
1998; Pape 1996
). Importantly, our study also
points out that some of the specific biophysical properties of
Ih in the stellate cells determine
that this current plays a major "pacemaker" role in the generation
of the theta-like subthreshold oscillations typical of these neurons
(Alonso and Llinás 1989
). Indeed, the combined
experimental and modeling analysis we carried out indicate that the
kinetic properties of both the activation and deactivation of
Ih implement a delayed feed-back
mechanism to the voltage changes led by a subthreshold
Na+ current that allows persistent subthreshold
oscillatory activity to occur. Although h currents have been
shown to contribute to rebound activity in many neuronal types
(Crepel and Penit-Soria 1986
; Mayer and Westbrook
1983
; Spain et al. 1987
) and to interact with
low-threshold Ca2+ currents (at rather negative
levels; about
70 mV) to generate oscillatory activity (Bal and
McCormick 1997
; Brown and DiFrancesco 1980
;
McCormick and Pape 1990
), to our knowledge, the SCs are the first case in which an h current has been shown to
generate persistent oscillatory activity by interacting with a
sustained Na+ current in the subthreshold voltage
range (about
55 mV). Intrinsic oscillatory activity in the
subthreshold voltage range may be of fundamental importance in defining
the integrative properties of the participating neurons (cf.
Hopfield 1995
; Lampl and Yarom 1993
;
Llinás 1988
).
A characteristic pharmacological feature of
Ih in all other cell types studied is
its blockade by Cs+ but not
Ba2+. Consistent with this,
Ih in the SCs was largely reduced,
though not completely blocked, by Cs+ (1-6 mM)
and not significantly affected by Ba2+ (1-2 mM).
The incomplete block of Ih by
Cs+, which also has been observed in other cells
(Champigny and Lenfant 1986; Ishii et al.
1999
), prompted us to use the recently described bradycardic
agent ZD7288 (BoSmith et al. 1993
). As found in other neurons (Gasparini and DiFrancesco 1997
; Harris
and Constanti 1995
; Lüthi et al. 1998
;
Maccaferri and McBain 1996
), ZD7288 fully and
selectively (in the voltage range explored:
45 to
120 mV) blocked
Ih in the SCs.
In general terms, the biophysical properties of
Ih as recorded in the SCs were similar
to those reported for other brain neurons though some differences in
activation threshold and reversal potential were apparent, and these
appear to be functionally significant. Ih in the SCs appeared as a
noninactivating inward current that turned on (activated) relatively
slowly with hyperpolarization and also turned off (deactivated) slowly
with depolarization. As with other voltage-dependent currents, the
conductance that gives rise to Ih
showed a sigmoidal activation curve with a threshold at around 45 mV,
a half-activation point at
77 mV, and a slope factor of 12.1. A
half-activation point in the range of
70 to
80 mV is typical of
other neurons; however, the Ih
activation threshold that we observed in the SCs was ~10-15 mV more
positive than that reported for other subcortical or cortical neurons
(Bayliss et al. 1994
; Crepel and Penit-Soria
1986
; Halliwell and Adams 1982
; Kamondi
and Reiner 1991
; Mayer and Westbrook 1983
;
McCormick and Pape 1990
; Mercuri et al.
1995
; Spain et al. 1987
). An activation threshold at about
45 mV is, however, comparable with that found in
cardiac sinoatrial cells and Purkinje fibers (DiFrancesco
1981
; DiFrancesco et al. 1986
; Yanagihara
and Irisawa 1980
) and to recent findings for hippocampal and
brain stem neurons (Maccaferri and McBain 1996
;
Maccaferri et al. 1993
; Travagli and Gillis
1994
). In addition, the estimated reversal potential for
Ih in the SCs (about
20 mV) was
10-30 mV more positive than that reported for other subcortical or
cortical neurons (Bayliss et al. 1994
; Crepel and
Penit-Soria 1986
; Halliwell and Adams 1982
;
Kamondi and Reiner 1991
; Mayer and Westbrook
1983
; McCormick and Pape 1990
; Mercuri et
al. 1995
; Spain et al. 1987
), though, again,
very similar to the value reported for cardiac sinoatrial cells and
Purkinje fibers (around
25 mV) (DiFrancesco 1981
;
DiFrancesco et al. 1986
; Yanagihara and Irisawa
1980
). A reversal potential around
20 mV clearly suggests
that Ih in the SCs, as in all other
excitable cells studied, is a mixed cationic current carried by both
Na+ and K+ ions. Consistent
with this interpretation, we found that raising [K+]o shifted
Vh in the depolarizing direction
(without any significant effect on the activation curve)
(Halliwell and Adams 1982
; Mayer and Westbrook
1983
; Spain et al. 1987
; Takahashi
1990
), whereas lowering
[Na+]o shifted
Vh in the hyperpolarizing direction.
Alterations in [Cl
]i
changed neither the reversal potential nor the activation curve corresponding to this current, suggesting that it is cation-specific.
With regard to the time-dependent properties of
Ih in the SCs, an interesting feature
was their biexponential kinetic nature. We found that the time course
of activation of Ih was described best
by a biexponential function having fast
(1) and slow
(
2) time constants that were voltage
dependent
decreasing with increasing hyperpolarization. A dual
exponential nature of the Ih
deactivation time course also was confirmed, with fast and slow time
constants that also showed voltage dependency
becoming faster with
increasing depolarization. Although many of the studies on
Ih report a time course of activation
that appears to be well fitted by a mono-exponential function
(Crepel and Penit-Soria 1986
; DiFrancesco et al.
1986
; Halliwell and Adams 1982
; McCormick
and Pape 1990
), some also have shown that a biexponential
fitting best represented the time course of
Ih in the corresponding cells
(Mayer and Westbrook 1983
; Solomon and Nerbonne
1993
; Spain et al. 1987
). The fact that the activation time constants reported in different neurons (measured at
similar temperatures) range from tens of milliseconds (Crepel and Penit-Soria 1986
), through hundreds of milliseconds
(Kamondi and Reiner 1991
) and even seconds
(McCormick and Pape 1990
; Soltesz et al.
1991
), might be interpreted as indicative of multiple
Ih channel subtypes with different
gating properties. Thus cells expressing more than one
Ih channel subtype would show multiple kinetic components during both the activation and deactivation processes. Strong evidence for this comes from a zebrafish mutant (slow mo), which exhibits a slower than normal heart rate. A
voltage-clamp analysis in isolated cardiac myocytes from the wild-type
and the mutant zebrafish demonstrated that
Ih was selectively decreased in the
mutant. Importantly, this decrease appeared to result from the
selective diminution of the fast kinetic component of the current
(Baker et al. 1997
). Finally, very recently, three
different groups have cloned various genes responsible for
Ih clearly demonstrating the existence
of a family of hyperpolarization-activated cation channels with
different activation kinetics (Gauss et al. 1998
; Ludwig et al. 1998
, 1999
; Santoro et al.
1998
).
Function of Ih in the stellate cells
In the stellate cells, the rather positive values that we found
for Ih activation (45 mV) and
reversal potential (
20 mV) indicate that in these neurons
Ih must be a major contributor to the
resting membrane potential (around
60 mV) (Alonso and Klink
1993
). Indeed, full block of
Ih with ZD7288 induced a robust hyperpolarization of the resting membrane potential in current-clamp conditions and a large outward shift in the holding current at
60 mV
in voltage-clamp conditions. In addition, in the stellate cells,
Ih also efficiently modulates membrane
hyperpolarization, as observed by the pronounced depolarizing sags in
response to membrane hyperpolarization and the robust rebound
potentials that follow. In this respect, the stellate cells are densely
innervated (Finch et al. 1988
; Jones
1994
; Jones and Buhl 1993
; Kohler
1988
; Kohler et al. 1985
; Wouterlood et
al. 1995
) by GABAergic inputs, and we have observed that
Ih efficiently limits the inhibitory postsynaptic potentials that these inputs trigger. Indeed
Ih activation in response to an
inhibitory postsynaptic potential implements a
"resetting" mechanism for the intrinsic oscillations (Alonso and
Dickson, unpublished observations).
As directly demonstrated in this study, a major role of Ih in the stellate cells is that of a "pacemaker" current. This role is not, however, one of providing a "background" depolarizing current that sustains rhythmic discharge but of contributing to the generation of persistent subthreshold oscillatory activity. We confirmed that the bradycardic agent ZD7288 selectively abolished Ih in the range of potentials corresponding to the oscillatory range and found that it progressively and completely abolished the rhythmic subthreshold oscillations. This evidence directly implicates Ih in their generation.
In the present study, we found no evidence of a slow outward
K+ conductance, such as the M current, being
necessary for the generation of the subthreshold oscillations by the
stellate cells. Indeed Ba2+, which blocks the
M-current as well as leak K+ conductances and
IKir, increased the amplitude of the
oscillations, which also became more regular and lower in frequency.
This result is in contrast to our previous study using sharp electrodes
(Alonso and Klink 1993) in which we reported an apparent
block of the oscillations with Ba2+. However, in
that study Ba2+ was applied in the absence of
glutamatergic and GABAergic neurotransmission and thus the apparent
block may have been due to the increased synaptic background activity
that is caused by Ba2+. In
addition, because Ba2+ also
prolonged the action potential, allowing for an increased Ca2+ influx and an enhancement of the slow
afterhyperpolarization (see Fig. 10 in Klink and Alonso
1993
), it could have been that the apparent block of the
oscillations also was related to a membrane shunting effect by a
Ca2+-dependent conductance. To test these
possibilities, we performed additional sharp electrodes recordings from
stellate cells in a similar manner as previously described
(Alonso and Klink 1993
) in which
Ba2+ application (1 mM) was conducted in the
presence of glutamatergic and GABAergic antagonists and/or the
Ca2+ channel blocker Co2+
(2 mM) (n = 6; not shown). In these experiments, in
agreement with the above interpretations, the application of
Ba2+ during synaptic and
Ca2+ conductance block with
Co2+ did not abolish the oscillations, which,
however, did become slower in frequency (as in our present whole cell
patch study). In addition, a similar result was observed when we
repeated the preceding experiment using perforated patch recordings
(Horn and Marty 1988
) (n = 2; not
shown). We did, however, observe that, using either sharp electrodes or
the perforated patch technique, block of Ca2+
conductances with Co2+ was required to best
reproduce our present results using conventional whole cell patch
recording. This suggests that a run-down of Ca2+
conductances occurred during the conventional whole cell technique that
allowed for a better expression of the oscillations in the presence of
Ba2+ (as seen in Fig. 9).
The modeling analysis that was carried out on the basis of the
biophysical properties of Ih and
INaP, as experimentally derived from
the stellate cells, indicated that the interplay between these two
currents appears sufficient for the generation of persistent subthreshold oscillations in these neurons. First, in the SCs, the
Gh activation curve has a rather
positive threshold for activation (at about 45 mV) and overlaps
substantially with the GNaP activation curve (threshold at about
65 mV) (Magistretti and Alonso
1999
), thus setting the stage for their potential interplay
because these conductances activate with voltage changes in opposite
directions. Second, and most importantly, the slow activation and
deactivation kinetics of Ih implement
a delayed feed-back mechanism to the voltage changes led by the
"instantaneous" changes in INaP,
thus causing the emergence of sustained oscillatory activity (given the
passive membrane properties of the cells). Alonso and
Llinás (1989)
initially had postulated an interaction
between Ih and INaP as the basis for the generation
of subthreshold oscillations by the SCs. Using a bifurcation analysis,
White et al. (1995)
also had implicated
Ih (in addition to an as-yet
undetermined slow outward rectifier) in the generation of these
oscillations. More recently, an interaction between
Ih and
INaP also has been proposed as the
basis for subthreshold membrane potential resonance in response to
oscillatory intracellular current injection in sensorimotor cortex
neurons (Hutcheon et al. 1996a
,b
). During the last 10 years, Na+-dependent subthreshold oscillations
similar to those expressed by the SCs also have been observed in other
brain neurons (Alonso et al. 1996
; Amitai
1994
; Gutfreund et al. 1995
; Leung and
Yim 1991
; Llinás et al. 1991
; Pape
et al. 1998
), and they have been considered typically to emerge
from the interplay between INaP and an
outward K+ current, such as an M-type current
(Gutfreund et al. 1995
; Llinás et al.
1991
; Pape et al. 1998
; White et al.
1995
, 1998
) or an A-type current (Wang 1993
). As
in other situations, distinct ionic mechanisms may be responsible for a
similar electrophysiological event in different neurons according to
their specific integrative needs.
Interestingly, we found that complete abolition of the rhythmic
subthreshold oscillations required a very substantial reduction of
Ih. Cs+, which
we found to reduce Ih by ~65%, did
not completely abolish the oscillations, thus suggesting that the
oscillatory process per se is very robust and rather insensitive to
variations in the level of Ih
expression. Indeed our simplified model maintained sustained
oscillatory activity even when Ih was
decreased by 50% (not shown). Further reductions caused a very rapidly
damping oscillation. However, in an elaborated SC model that we have
constructed (Fransén et al. 1998) when the impact
of channel noise (White et al. 1998
) is considered, the
SC model behaved much more closely to the "real" cell in that
Ih reductions of up to ~70% were
required to completely abolish subthreshold rhythmicity. The critical
importance of stochastic noise in sculpturing the membrane voltage
behavior of the SCs has been treated in detail by White et al.
(1998)
.
Functional role of the subthreshold oscillations
The potential functional role of subthreshold oscillatory activity
in neocortical neurons was reviewed recently by Connors (Connors
and Amitai 1997) and also has been treated in detail by others
(Engel et al. 1992
; Gray 1994
;
Laurent 1996
; Singer 1993
). We will
discuss briefly here the potential functional implications that the
presence of rhythmic subthreshold oscillatory activity in the stellate
cells may have with regard to temporal lobe function. A
well-established role of the entorhinal network is that of memory function. Indeed the stellate cells from EC layer II occupy a privileged position in the neocortico-hippocampo-neocortical circuit. They are the targets of convergent information from polysensory associational areas that they funnel to the dentate gyrus of the hippocampal formation via the perforant path. Thus a potential role of
the intrinsic oscillatory activity of the stellate cells could be that
of implementing a synchronizing mechanism by which convergent sensory
information is coordinated temporally for its transfer to the
hippocampal processing machinery and the formation of a memory event
(Buzsáki 1989
, 1996
; Lopes da Silva et al. 1985
; Rudell et al. 1980
; ). Although still far
from directly testing such a hypothesis given the complex nature of the
information processed by the EC, we do know that EC layer II neurons
are powerful generators of theta rhythmicity "in vivo"
(Alonso and García-Austt 1987a
,b
; Dickson
et al. 1995
; Mitchell and Ranck 1980
), and the theta rhythm has been implicated in memory processes (Greenstein et al. 1988
; Holscher et al. 1997
; Huerta
and Lisman 1993
; Larson et al. 1986
;
Lisman and Idiart 1995
; Pavlides et al.
1988
; Winson 1978
). Importantly, analysis of
inhibitory synaptic input on EC layer II neurons show that inhibitory
postsynaptic potentials, which EC stellates are likely to receive
during theta (cf. Fox 1989
; Leung and Yim
1986
; Soltesz and Deschênes 1993
;
Ylinen et al. 1995
) can very efficiently reset the
intrinsic oscillations (Alonso and Dickson, unpublished observations;
see also Cobb et al. 1995
). In addition, theoretical,
experimental and modeling studies also indicate that the intrinsic
subthreshold oscillations of individual cells can engender synchronized
population activity (Engel et al. 1992
;
Fransén et al. 1999
; Jefferys et al.
1996
; König et al. 1995
; Lampl and
Yarom 1993
; Singer 1993
; Steriade et al.
1990
).
In conclusion, the stellate cells from EC layer II display a very robust Ih current, which, in conjunction with INaP, gives rise to the expression of rhythmic subthreshold oscillations of the membrane potential in these cells. These oscillations are likely to implement a synchronizing mechanism that may be important in the memory function of the temporal lobe. Understanding the biophysical/molecular and neuromodulatory aspects of the distinct voltage-gated ion channels underlying Ih and INaP thus may be of critical importance in exploring the functions of the EC network.
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ACKNOWLEDGMENTS |
---|
We thank I. Manns for kind and efficient aid in Matlab programming.
This study was funded by grants from the Fonds de la Recherche en Santé du Québec and the Medical Research Council (MRC) to A. Alonso and a grant from the Human Frontier Science Program Organization (HFSP) to A. Alonso and M. Hasselmo. C. T. Dickson was supported by fellowships from the National Sciences and Engineering Research Council and the Savoy Foundation. M. H. Shalinsky was supported by a studentship from the MRC. E. Fransén was supported by the Swedish Foundation for International Cooperation in Research in Higher Education (STINT) and the P. E. Lindahls fund.
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FOOTNOTES |
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Address for reprint requests: A. Alonso, Dept. of Neurology and Neurosurgery, McGill University, 3801 University St., Montreal, Quebec H3A 2B4, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 August 1999; accepted in final form 11 January 2000.
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REFERENCES |
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