Institut für Neurophysiologie, Heinrich-Heine-Universität Düsseldorf, D-40001 Düsseldorf, Germany
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ABSTRACT |
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Kilb, Werner and
Heiko J. Luhmann.
Characterization of a Hyperpolarization-Activated Inward Current
in Cajal-Retzius Cells in Rat Neonatal Neocortex.
J. Neurophysiol. 84: 1681-1691, 2000.
Cajal-Retzius
cells are among the first neurons appearing during corticogenesis and
play an important role in the establishment of cortical lamination. To
characterize the hyperpolarization-activated inward current
(Ih) and to investigate whether
Ih contributes to the relatively positive
resting membrane potential (RMP) of these cells, we analyzed the
properties of Ih in visually identified Cajal-Retzius cells in cortical slices from neonatal rats using the
whole cell patch-clamp technique. Membrane hyperpolarization to 90 mV
activated a prominent inward current that was inhibited by 1 mM
Cs+ and was insensitive to 1 mM Ba2+. The
activation time constant for Ih was strongly
voltage dependent. In Na+-free solution,
Ih was reduced, indicating a contribution of
Na+. An analysis of the tail currents revealed a reversal
potential of
45.2 mV, corresponding to a permeability coefficient
(pNa+/pK+) of 0.13. While an increase in the
extracellular K+ concentration
([K+]e) enhances
Ih, it was reduced by a
[K+]e decrease. This
[K+]e dependence could not be explained by an
effect on the electromotive force on K+ but suggested an
additional extracellular binding site for K+ with an
apparent dissociation constant of 7.2 mM. Complete Cl
substitution by Br
, I
, or
NO3
had no significant effect on
Ih, whereas a complete Cl
substitution by the organic compounds methylsulfate, isethionate, or
gluconate reduced Ih by ~40%. The
Ih reduction observed in gluconate could be
abolished by the addition of Cl
. The analysis of the
[Cl
]e dependence of
Ih revealed a dissociation constant of 9.8 mM and a Hill-coefficient of 2.5, while the assumption of a
gluconate-dependent Ih reduction required an
unreasonably high Hill-coefficient >20. An internal perfusion with the
lidocaine derivative lidocaine N-ethyl bromide blocks
Ih within 1 min after establishment of the
whole cell configuration. An inhibition of
Ih by 1 mM Cs+ was without an
effect on RMP, action potential amplitude, threshold, width, or
afterhyperpolarization. We conclude from these results that
Cajal-Retzius cells express a prominent Ih
with characteristic properties that does not contribute to the RMP.
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INTRODUCTION |
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A delayed inward
current that is activated by membrane hyperpolarization has been found
and characterized in cardiac cells (DiFrancesco 1986;
Yanagihara and Irisawa 1980
) and in a variety of
neuronal cells including photoreceptors, hippocampal CA1 pyramidal cells, thalamic relay, spinal cord, and neocortical neurons
(Hestrin 1987
; Maccaferri et al. 1993
;
Mayer and Westbrook 1983
; Pape and McCormick
1989
; Solomon and Nerbonne 1993a
). For
neuronal cells, this current was termed
Ih, for hyperpolarization-activated
current (Pape 1996
). The
Ih is carried by the monovalent
cations Na+ and K+, showed
a delayed onset and a slow activation, and could be blocked by
extracellular Cs+ but not by
Ba2+ (reviewed in Pape 1996
). In
neuronal cells, Ih was thought to be
involved in rhythm generation and in the determination of resting membrane potential (Maccaferri et al. 1993
;
McCormick and Pape 1990a
; Soltesz et al.
1991
). Four different ion channels related to these currents
were recently identified (Ludwig et al. 1998
; Santoro et al. 1998
, Seifert et al.
1999
).
The existence of a hyperpolarization-activated inward current has
also been suggested in Cajal-Retzius cells because these cells show a
hyperpolarization-activated voltage sag (Zhou and Hablitz
1996a). Cajal-Retzius cells are thought to be the first cell
type appearing in the developing neocortex (Bayer and Altman 1991
). They play an important role in the establishment of the cortical lamination (for review, see Frotscher 1998
).
Most, if not all, of the Cajal-Retzius cells disappear later in
development most probably by apoptosis (Derer and Derer
1990
; Meyer and Gonzales-Hernandez 1993
;
Naqui et al. 1999
; but see Martin et al.
1999
; Parnavelas and Edmunds 1983
).
Cajal-Retzius cells display the typical electrophysiological properties
of immature neurons: they have a relatively positive resting membrane
potential (RMP), a high input resistance, and slow action potentials
with a high threshold (Hestrin and Armstrong 1996
;
Zhou and Hablitz 1996a
). The relatively positive RMP may be involved in the susceptibility of Cajal-Retzius neurons for neuronal
cell death (Mienville and Pesold 1999
).
Because hyperpolarization-activated inward currents were thought to play an important role in the determination of RMP, we analyzed the properties of the hyperpolarization-activated inward current in Cajal-Retzius cells in detail to elucidate the physiological relevance of these currents. We demonstrate that Cajal-Retzius cells express a prominent hyperpolarization-activated inward current that resembles the properties of Ih and that this current does not contribute to the high RMP of these cells. In addition we show for the first time that the neuronal Ih is regulated by extracellular small anions.
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METHODS |
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Slice preparation
Neonatal Wistar rats (postnatal day 0-3) were deeply
anesthetized by hypothermia and decapitated. The brain was quickly
removed and stored for 1-2 min in ice-cold artificial cerebrospinal
fluid (ACSF). The hemispheres were dissected at the midline, and the pia was removed carefully, using fine tweezers. Tangential neocortical slices (maximum thickness, 400 µm) of both hemispheres were cut on a
vibratome (Pelco 101, TPI, St. Louis, MO) using a purpose build holder,
which allows to take tangential slices from various regions of the
neocortex. The slices were subsequently mounted on fine tissue paper
(Kodak Lens Paper) to enable the reconstruction of their original
orientation in the neocortex and were transferred to an incubation
chamber filled with equilibrated ACSF at 32°C in which they recovered
for 1 h before recording began.
Identification of cells
The cells of the superficial layer of the tangential neocortical
slices were visualized using infrared DIC-videomicroscopy (Dodt
and Zieglgänsberger 1990). Cajal-Retzius cells were
identified by their unique morphology and electrophysiological
properties. Only cells located near the surface of the slice with an
unambiguous root like appearance and one thick tapered process (see
Fig. 1A) were
chosen for experimental examination. Cells were excluded from analysis
if their electrophysiological properties did not fit the results
reported for Cajal-Retzius cells (RMP between
35 and
75 mV, input
resistance >600 M
, slow action potentials with prominent
afterhyperpolarization, action potential duration at half-maximal
amplitude >5 ms; compare to Hestrin and Armstrong 1996
;
Zhou and Hablitz 1996a
).
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Experimental setup and procedure
The DIC-videomicroscopic setup consisted of an upright
microscope with differential interference contrast optics (Axioskop, Zeiss, Jena, Germany), an infrared filter (KMZ 50-2,
1/2 = 750 nm, width 57 nm, Schott, Mainz,
Germany), and a CCD-camera (C5405, Hamamatsu, Japan). The video image
was contrast enhanced by a video processor (C 2400, Hamamatsu),
visualized on a video-monitor, and digitized on-line using a frame
grabber card (Screen machine II, Fast, Munich, Germany).
Whole cell patch-clamp recordings were performed according to the
procedure described by Stuart et al. (1993). Patch
pipettes were pulled from borosillicate tubing (2.0-mm OD, 1.16-mm ID; Science Products, Hofheim, Germany) using a vertical puller (PP-83, Narishige, Tokyo). The patch pipettes were connected to the
headstage of a discontinuous voltage-clamp/current-clamp amplifier
(SEC05L, NPI, Tamm, Germany). Signals were amplified, low-pass filtered at 3 kHz, visualized on an oscilloscope (TDS210, Tektronix, Beaverton, OR), digitized on-line by an AD/DA-board (ITC-16, Heka,
Lamprecht, Germany), recorded and processed with the software
WINTIDA 4.11 (Heka), and stored on a personal computer. The
bathing solution was connected to ground via a chlorided silver wire
except for all Cl
substitution experiments,
where a agar bridge (3 M KCl/5% agar) was used. The agar bridge
reduced the potential changes induced by the
Cl
-substitutes to <3 mV. The agar bridge was
also used to determine the liquid junction potential of the
gluconate-based pipette solution, which amounted to 9.6 ± 2.6 mV
(mean ± SD; n = 7).
The slices were transferred into a submerged recording chamber (volume
ca. 1 ml) mounted on the fixed stage of the microscope and were
superfused with ACSF at a rate of 1-2 ml/min. All experiments were
performed at 32°C. Gentle pressure was applied to advance the
electrode to the surface of the cell. After obtaining a stable seal of
>1 G, the seal was broken by suction. As soon as the whole cell
configuration was established, the RMP was recorded and the intrinsic
membrane properties were analyzed under current-clamp conditions. For
the determination of the input resistance, the current voltage
relation, and the active membrane properties, hyperpolarizing and
depolarizing current pulses were injected from a holding potential of
60 mV. The input resistance was calculated from a membrane
hyperpolarization induced by a current pulse according to Ohm's law.
The spike amplitude was measured from the spike threshold and the spike
width was determined at the half-maximal spike amplitude.
Histochemical procedure
In all experiments 0.5% biocytin (Sigma, Deissenhofen, Germany)
was added to the pipette solution to label the cells on which a whole
cell configuration was established. To stain the labeled cells, slices
were processed by a modification of the technique described by
Horikawa and Armstrong (1988). Slices were fixed in a
4% paraformaldehyde solution for
24 h subsequently to the experiment, rinsed, and were incubated 60 min with 0.5%
H2O2 and 0.8% Triton-X to
inhibit endogenous peroxidases. An overnight incubation with an
avidin-coupled peroxidase (ABC kit, Vectorlabs, Burlingame, CA) was
followed by a preincubation in 0.5 mM diaminobenzidine and a subsequent
reaction in diaminobenzidine and 0.015%
H2O2. Staining was
intensified by a treatment with 0.15% OsO4. The
slice was then rinsed, dehydrated slowly through alcohol and
propylenoxide, and embedded in Durcopan (Fluka, Buchs, Switzerland).
Solutions
ACSF consisted of (in mM) 124 NaCl, 26 NaHCO3, 1.25 NaH2PO5, 1.8 MgCl2, 1.6 CaCl2, 3 KCl,
and 20 glucose and was equilibrated with 95%
O2-5% CO2. The pH of this
solution was 7.4 pH units and the osmolarity was 336 mOsm. In
experiments in which the membrane was depolarized to potentials above
30 mV, 1 µM tetrodotoxin (TTX), 20 mM tetraethylammonium (TEA), 6 mM 4-aminopyridine (4-AP), 300 µM Cd2+, and 100 µM Ni2+ were added to the bathing solution to
block voltage-gated Na+ currents,
K+ currents, and low- and high-voltage-activated
Ca2+ currents, respectively. TEA,
Ni2+, Cd2+,
Cs+, and Ba2+ were added to
the saline solutions as chloride salts, Ni2+,
Cd2+, Cs+, and
Ba2+ without osmotic compensation, while TEA
substituted 20 mM Na+. In
Na+-free solutions, NaCl and
NaHCO3 were replaced by an equimolar amount of
choline chloride and choline bicarbonate, respectively, while altered
extracellular K+ concentrations
([K+]e) were compensated
by variation of the extracellular Na+
concentration ([Na+]e).
In Cl
-free solutions,
Cl
was substituted with
I
, Br
,
NO3
, gluconate, isethionate, or
methylsulfate. In detail these solutions consisted of (in mM)
Br
substituted: 124 NaBr, 3 KBr, 1.6 CaBr2, 1.8 MgBr2;
I
substituted: 124 NaI, 3 KI, 1.6 CaBr2, 1.8 MgBr2;
NO3
substituted:
124 NaNO3, 3 KNO3, 1.6 Ca(NO3)2, 1.8 Mg(NO3)2; gluconate substituted: 124 Na-gluconate, 3 K-gluconate, 1.6 Ca-gluconate, 1.8 Mg-gluconate; Isethionate substituted: 124 Na-isethionate, 3 K-gluconate, 1.6 Ca-gluconate, 1.8 Mg-gluconate; and methylsulfate substituted: 124 Na-methylsulfate, 3 K-methylsulfate, 1.6 Ca-gluconate, 1.8 Mg-gluconate. Different extracellular Cl
concentrations ([Cl
]e)
were obtained by adding ACSF to gluconate-substituted
Cl
-free solution. The pipette solution
contained (in mM) 117 K-gluconate, 13 KCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 2 Na2-ATP, and 0.5 Na-GTP, pH adjusted to
7.4 with KOH and osmolarity to 306 mOsm with sucrose. In some
experiments, 5 mM lidocaine N-ethyl bromide (QX-314) was
added to the pipette solution. QX-314 and TTX were purchased from RBI
(Natic, MA), Na-isethionate and all nitrate salts from Fluka,
Na-methylsulfate, MgBr2, and
CaBr2 from Aldrich (Milwaukee, WI),
K-methylsulfate from ICN (Aurora, OH), and all other substances from Sigma.
Statistics
If not otherwise noted, all values were expressed as means ± SD. For statistical analysis the Student's t-test was used, results were designated significant at a level of P < 0.05.
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RESULTS |
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Properties of the examined cells
Cajal-Retzius cells (n = 109) from 33 animals were
used for this investigation. Thirty-six of the 109 investigated cells
could be stained with biocytin. In the other cases, the cells were
probably removed from the slice because they were detached with the
electrode upon withdrawal (compare to Hestrin and Armstrong
1996; Zhou and Hablitz 1996a
). All of these 36 stained cells showed the typical morphological features of
Cajal-Retzius cells: an ovoid soma, a thick tapered dendrite, and
poorly ramified dendritic processes (Fig. 1B).
The mean RMP of the Cajal-Retzius cells was 45.8 ± 7.7 mV
(n = 109), and the mean input resistance was 1,367 ± 510 M
(n = 109). The cells elicited action
potentials at a threshold of
28.4 ± 10.8 mV (n = 94) and with an amplitude of 35.7 ± 12.6 mV if depolarizing
currents were applied (Fig. 1C). The first action potential
had a width at half-maximal spike amplitude of 10.1 ± 5.3 ms,
while a distinct action potential broadening during repetitive spiking
could be observed.
Hyperpolarization activates a Cs+-sensitive voltage sag
Figure 1C also demonstrates the characteristic response
of a Cajal-Retzius cell to the injection of hyperpolarizing currents (Zhou and Hablitz 1996a). After the membrane was
hyperpolarized to below
90 mV, an obvious sag in the membrane
potential (Em) was observed. The
amplitude of this voltage sag depended on the maximal amplitude of the
hyperpolarization (Fig. 1, C and D). The
hyperpolarization-activated voltage sag was reversibly blocked by
extracellular application of 1 mM Cs+
(n = 8, Fig.
2A) while extracellular
application of 1 mM Ba2+ had no effect on the
voltage sag (n = 8, Fig. 2B), although
Ba2+ led to a membrane depolarization of
13.6 ± 7.4 mV (n = 8) and increased the input
resistance by 121 ± 22% (n = 8). The
depolarizing effect of Ba2+ in combination with
the observed increase in the input resistance may suggest an inhibitory
effect of Ba2+ on a tonically active
K+ conductance (Sutor and Hablitz
1993
). The decline time of the voltage sag appeared to be
voltage dependent (see Fig. 1C), which made the analysis of
the underlying inward currents complex if Em was not constant. Thus for further
analysis voltage-clamp experiments were performed.
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The voltage sag is due to a slowly activating Cs+-sensitive inward current
Figure 3A shows a typical
voltage-clamp recording of the hyperpolarization-activated inward
current. A membrane hyperpolarization below a threshold potential of
about 90 mV induced an additional component of the inward current
that could be distinguished by its slow activation. Corresponding to
the observations in the current-clamp experiments, the application of 1 mM Ba2+ had no significant effect on the slowly
activating inward current (n = 8, Fig. 3B),
while the application of 1 mM Cs+
(n = 8) caused a complete block of the slowly
activating component of the inward current, without affecting the
capacitive and the "leakage" (= Cs+
insensitive) components (Fig. 3C).
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For a first analysis, we investigated the
Cs+-sensitive component of the inward current
(Fig. 3D), which was obtained by subtracting the current
traces recorded in the presence of 1 mM Cs+ from
the control traces. If the amplitude of the
Cs+-sensitive component was plotted against
Em (Fig. 3E), the data could be fitted with the Boltzmann equation
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(1) |
To facilitate the analysis of the current, the slowly activating
component of the inward current (Is)
was isolated from the leakage current (or instantaneous current = Ii) by fitting the current trace with
an exponential function (see Fig.
4A), a method also used by
Perkins and Wong (1995). The current traces of all experiments could be fitted with a single exponential function. The
isolated Is component displayed a
nearly linear current-voltage relation between
100 and
130 mV,
activated at
89.0 mV (Fig. 4B) and disappeared in the
presence of 1 mM Cs+ (n = 19, data not shown). The plotted data could be fitted by a Boltzmann
equation using an Imax of
68 pA, an
E1/2 of
118 mV, and a s
of 14.8 mV. The activation time constant showed a strong voltage
dependence with a slope of 18.4 ms/mV (Fig. 4C). Because
these results were similar to the results obtained from the
Cs+-sensitive current component, the method of
Perkins and Wong (1995)
was used for the further
analysis of the hyperpolarization-activated inward current.
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To examine whether the hyperpolarization-activated inward current
depends on the basal Em, a 30-s
lasting depolarization to 0 mV was applied prior to the hyperpolarizing
steps (Fig. 4D). The solution used for these experiments
contained 1 µM TTX, 20 mM TEA, 6 mM 4AP, 300 µM
Cd2+, and 100 µM Ni2+ to
prevent the activation of voltage gated Na+,
K+, and Ca2+ currents
during the depolarization. The predepolarization to 0 mV
(n = 4) shifted the threshold potential of the
Is component (75.8 mV) and
E1/2 (
109.0 mV) to more positive
potentials (Fig. 4E). In addition, we analyzed very slow
activation kinetics of the Is
component at supra- and subthreshold potentials by investigating the
time course of current activation for
30 s. However, we could not
find an additional slowly activating component of the current (n = 7; data not shown). Furthermore, it had been shown
that E1/2 critically depends on
complete current activation, which requires longer hyperpolarized
pulses at more positive potentials due to the strong time dependence of
the activation kinetic (Seifert et al. 1999
). Thus we
examined the effect of an increased length of the hyperpolarizing pulse
on E1/2. Increasing the pulse length from 2 to 5 s shifted E1/2 to
105 mV.
Ionic nature of the slowly activating inward current
For the analysis of an influence of
[Na+]e on the
Is component of the inward current,
Na+ was completely removed from the bathing
solution. Figure 5A shows that
under these conditions, the Is
component was reduced by 70.4 ± 12.6% (n = 6;
analyzed at 120 mV), while the Ii
component was not significantly affected (112.8 ± 17.6%).
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To determine the reversal potential of the
Is component, an analysis of the tail
currents was necessary because it activates at potentials negative to
the expected reversal potential of a mixed cation current (Pape
1996). To enable the examination of the reversal potential,
voltage-gated Na+, K+, and
Ca2+ currents were blocked by 1 µM TTX, 20 mM
TEA, 6 mM 4-AP, 300 µM Cd2+, and 100 µM
Ni2+ during the experiments. A characteristic
experiment is shown in Fig. 5B. The membrane was
hyperpolarized to
110 mV for 2 s to activate the slowly
activating inward current and subsequently depolarized to potentials
ranging from
60 to 0 mV. For the analysis of the tail currents, an
exponential function was fitted to the current traces, using a time
frame from 0.2 until 2 s after the depolarizing step, to avoid
contaminations by capacitive currents. A single-exponential function
was sufficient to fit the tail currents. The exponential curve was
extrapolated to the beginning of the depolarizing step and this value
was taken as the expected maximal amplitude of the tail current
(It0) (see Fig. 5B, inset).
In Fig. 5C, this It0 was
plotted against the Em. The
current-voltage relation of the tail currents was linear in the
examined voltage range and reversed at an
Em of
45.2 mV. The time constants
for the inactivation of the tail currents showed no significant
dependence from Em.
The slowly activating inward current is affected by extracellular K+
It has been reported that
[K+]e affects the
hyperpolarization-activated inward current (Edman and Grampp
1989; Hestrin 1987
; Maccaferri et al.
1993
; McCormick and Pape 1990a
; Solomon
and Nerbonne 1993a
). Thus we tested the effect of lowered and
raised [K+]e on the
Ii and
Is components of the
hyperpolarization-activated inward current. A typical experiment in
K+-free saline solution is shown in Fig.
6A. The
Is component of the inward current was
nearly completely abolished under these conditions (9.1 ± 7.0%;
n = 6; analyzed at
120 mV), while the Ii component was not significantly
affected. A [K+]e
increase to 6, 8, 16, and 20 mM enhanced the
Is component, while it was decreased
by a [K+]e reduction to 2 mM (Fig. 6B). Boltzmann fits of the data revealed that
[K+]e affected only
Imax and had no significant effect on
E1/2 and s (Table
1). Neither a
[K+]e increase nor a
[K+]e decrease had a
significant effect on the time constant of the Is component or on the
Ii component. For a quantitative
analysis of the relation between Is
and [K+]e, the cord
conductance (gh = Is/Em;
analyzed at
120 mV) was plotted against
[K+]e in a
double-reciprocal diagram (Fig. 6C) and fitted by linear regression to obtain the maximal gh
(gmax) and the apparent dissociation constant for K+
(Kapp). From this plot, a
gmax of 1.1 fS and a
Kapp of 7.2 mM were calculated. If
Is was plotted versus
[K+]e (Fig.
6D) the data could be fitted using a Michaelis-Menten-like function
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(2) |
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(3) |
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The slowly activating inward current is affected by
extracellular Cl
Previous investigations demonstrated that the
hyperpolarization-activated inward current declined in
Cl-reduced solutions (Frace et al.
1992
; Mayer and Westbrook 1983
; McCormick
and Pape 1990a
; Yanagihara and Irisawa 1980
).
While it was proposed for neuronal cells that this decline in
[Cl
]e-reduced solutions
was caused by a blocking action of the Cl
substitutes (Mayer and Westbrook 1983
; McCormick
and Pape 1990a
), Frace et al. (1992)
suggested
for sino-atrial node cells that the hyperpolarization-activated inward
current depends directly on small anions. Thus we tested the effect of
various organic and inorganic substances as Cl
substitutes on the Ii and
Is component of the
hyperpolarization-activated inward current. A characteristic experiment
is shown in Fig. 7A. The
inorganic Cl
substitutes
Br
(122 ± 7%, n = 7),
I
(130 ± 11%, n = 8),
and NO3
(112 ± 5%,
n = 5) had no significant effect on the amplitude of
the hyperpolarization-activated inward current. The organic substitutes isethionate (n = 11), methylsulfate
(n = 9), and gluconate (n = 5) caused a
significant decrease in the Is
component by 38 ± 5, 41 ± 10, and 42 ± 8%,
respectively, while the time constant and the
Ii component were not significantly
altered. The Boltzmann fits of these data (Fig. 7B) revealed
that the Is reduction was caused by a
reduction in Imax, while
E1/2 or s were not
affected.
|
To investigate whether the effect of Cl
substitution was caused by a blocking action of the organic anions, we
examined the effect of different
[Cl
]e on the
Is component. In these experiments,
Cl
was substituted for gluconate. Figure
7C shows that a
[Cl
]e increase led to a
recovery of the Is component. The
[Cl
]e dependence of the
Is component could be fitted using a
Hill-like function (Fig. 7C)
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(4) |
The slowly activating inward current is blocked by intracellular QX-314
To test whether the slowly activating inward current was blocked
by intracellular administration of the lidocaine derivative QX-314
(Perkins and Wong 1995), we used an electrode solution containing 5 mM QX-314. A depolarizing step from
60 to
110 mV was
applied immediately (8 s) after the establishment of the whole cell
configuration and was repeated every 10 s for up to 4 min. While
the Ii component was not affected
during this experiment, the amplitude of the
Is component declined within 40 s
to <10% (n = 6) compared to the first hyperpolarizing
pulse (Fig. 8). Because the analysis of
the QX-314 effect required an immediate start of hyperpolarizing
pulses, these six cells could not be tested for action potential
properties and were thus identified only by their morphological and
passive electrophysiological characteristics.
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The slowly activating inward current has no effect on RMP and action potential properties
To determine whether the hyperpolarization-activated inward
current contributes to the RMP of Cajal-Retzius cells, we tested the
effect of 1 mM Cs+ on the RMP of these cells.
After a 4-min incubation with Cs+, no significant
difference in the RMP was observed (47.0 ± 2.6 mV vs.
46.5 ± 2.0 mV, n = 10). In addition we examined
the effect of Cs+ on action potential properties.
The application of 1 mM Cs+ had no effect on
action potential amplitude, threshold, width, and frequency or on the
amplitude of the afterhyperpolarization (n = 7, data
not shown).
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DISCUSSION |
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Identification of Cajal-Retzius cells
Cajal-Retzius cells possess an unique morphological appearance,
which makes it easy to identify them visually. In addition, all cells
appearing in this investigation displayed electrophysiological properties typical for Cajal-Retzius cells. They had a relatively positive RMP, had a high input resistance, and showed action potentials of small amplitude and slow time course that were elicited at a low
threshold and expressed a pronounced broadening (Hestrin and
Armstrong 1996; Mienville and Pesold 1999
;
Zhou and Hablitz 1996a
). All 36 histologically recovered
cells showed the morphological features typical for Cajal-Retzius
cells, such as an ovoid soma, one thick, tapered dendrite, and sparsely
branched dendritic processes (Hestrin and Armstrong
1996
; Zhou and Hablitz 1996b
). This finding demonstrates that the identification of Cajal-Retzius cells by their
appearance in the infrared videomicroscope in combination with their
electrophysiological properties is sufficient for an unambiguous identification.
Properties of the slowly activating inward current
For a characterization of the slowly activating inward current, it
is important to demonstrate that it is not caused by a shift in driving
forces evoked by ion accumulation or depletion but that it relates to
the direct activation of a membrane conductance (Pape
1996). The findings that the inward current was well described with the Boltzmann equation, expressed a strong
[K+]e dependence, which
cannot be explained by an effect on the driving force of
K+ ions, and could be selectively blocked
strongly suggest a conventional membrane conductance as the physical
correlate of this current. Our results clearly confirm that the slowly
activating inward current examined in this investigation resembles the
characteristic properties for Ih
(Pape 1996
). It showed a slow activation after a
hyperpolarizing step, depended on extracellular
K+ and Na+, and was blocked
by intracellular QX-314 and extracellular Cs+ but
was not affected by 1 mM Ba2+. Thus we can refer
to the slowly activating inward current of Cajal-Retzius cells as
Ih.
The activation of Ih could be well
described by a single-exponential function, resembling previous reports
(but see Solomon and Nerbonne 1993b). The activation
time constant showed a strong voltage dependence and was in the range
found in other examinations (McCormick and Pape 1990a
;
Santoro et al. 1998
; Solomon and Nerbonne 1993b
). On the other hand, Ih
activated at more negative Em than reported for other preparations (Ludwig et al. 1998
;
Maccaferri et al. 1993
; Mayer and Westbrook
1983
; Pape and McCormick 1989
; Santoro et
al. 1998
; Solomon and Nerbonne 1993a
;
Wollmuth 1995
). The negative activation threshold of
Ih was puzzling because the RMP of
Cajal-Retzius cells is more positive as compared with the cells in
these reports. This discrepancy between the RMP and the activation
threshold was not effected by errors in the determination of these
potentials due to liquid-junction potentials because the measured
liquid junction potential would shift both values by ~10 mV toward
positive direction. It had been shown by Seifert et al.
(1999)
that the Ih current
amplitude at more positive potentials might be underestimated if the
hyperpolarizing pulse was too short because
Ih may not reach steady-state values
at positive potentials due to the strong voltage dependence of the
Ih activation kinetic. Thus the
estimation of E1/2 critically depends
on the length of the hyperpolarizing pulse. In our investigation, an
attenuation of the hyperpolarizing pulse to 5 s indeed shifted
E1/2 by 13 mV in positive direction.
However, even under these conditions, the activation threshold of
Ih was far more negative than the RMP
of Cajal-Retzius cells. These findings suggests that
Ih was not activated under resting
conditions and thus did not contribute to the maintenance of the RMP.
This suggestion was confirmed by the finding that the RMP was
unaffected by a block of Ih with 1 mM
Cs+.
To test whether Ih was partially
inactivated at a holding potential of 60 mV, the
Ih activation threshold was determined after a 30-s lasting depolarization to 0 mV. This massive
depolarization prior to the hyperpolarizing step had only a minor
effect on Ih activation threshold,
which was still far more negative than the RMP. Thus
Ih could not be activated near RMP
even under these conditions. If the observed shift in activation
threshold was due to the release of inactivation, a mechanism never
reported for Ih (Pape
1996
), or due to shifts in driving forces evoked by ion
accumulation or depletion could not be answered unequivocally from our observation.
Ionic nature of the inward current
Due to the equilibration of the cell interior with the pipette
solution occurring in whole cell mode the intra- as well as the
extracellular concentrations of Na+ and
K+ are known. Thus it is possible to estimate the
ion permeabilities of Ih without
analyzing shifts in the reversal potential caused by alterations in
extracellular ion concentrations. From the determined reversal
potential of 45.2 mV a permeability coefficient (
= pNa+/pK+) of 0.13 was
calculated using the Goldman equation (Hille 1984
). This
value for
was in the range reported for other neuronal preparations
(
= 0.24, Ludwig et al. 1998
; 0.25, Hestrin 1987
; 0.40, Solomon and Nerbonne
1993a
) and demonstrates that
Ih is a mixed
Na+/K+ current in
Cajal-Retzius neurons. We observed that complete substitution of
Na+ with choline caused an incomplete block of
Ih. A fraction of ~30% (analyzed at
120 mV) preserved under this conditions. This remaining fraction
cannot be explained sufficiently by a K+ influx
due to the inwardly directed electromotive force on
K+ under these conditions because we estimate
from the Goldman equation that only 3% of
Ih should maintain in
Na+-free solution. This finding may be an
indication that the Na+ influx interferes with
K+ fluxes by the same mechanisms as described by
Wollmuth (1995)
for the inhibiting action of
submillimolar [Na+]e.
The complete omission of K+ from the
extracellular solution blocked Ih
nearly completely, a finding that could not be explained by the effect
of [K+]e on driving
forces. Thus an alternative explanation for this effect must be
considered. Alterations in
[K+]e led to massive
shifts in Ih amplitude that also could
not sufficiently be explained by the effect of
[K+]e on the driving
force. However, the observed relation between Ih and
[K+]e could be modeled
with a Michaelis-Menten-like equation, suggesting an additional
regulatory binding site for K+. The calculated
Kapp for K+ of
7.2 mM is lower than reported for other preparations (20 mM, Edman and Grampp 1989; 25.7 mM, Solomon and
Nerbonne 1993a
). The observed relation between
[K+]e and
Ih predicts relatively great shifts
in Ih evoked by small [K+]e alterations in the
physiological [K+]e
range. Thus in Cajal-Retzius cells, Ih
may act as a sensor for
[K+]e, which may be
influenced by various physiological and pathophysiological events
(Coles and Poulain 1991
; Perez-Pinzon et al.
1995
).
The complete Cl substitution with the organic
anions isethionate, methylsulfate, or gluconate evoked a significant
reduction in Ih that could be
abolished by the readdition of Cl
. Our results
argue against the suggestion that this reduction was caused by a
blocking action of the organic substitute itself (Mayer and
Westbrook 1983
; McCormick and Pape 1990a
). We
observed an almost identical blocking effect for all organic compounds used in this investigation despite their different molecular structure. We also observed that the analysis of the
[Cl
]e dependence of
Ih reduction demonstrates that an
unreasonable high Hill coefficient >20 is required, if a blocking
action of gluconate is assumed. Thus we suggest that the
Cl
anions itself modulate
Ih. Corresponding to investigations on cardiac cells (Frace et al. 1992
), we observed that
substitution of Cl
with the small inorganic
anions Br
, I
, or
NO3
had no significant effect
on Ih. This result suggests that at least some small anions can replace Cl
in
Ih modulation. Although no complete
block of Ih could be observed in
Cl
-free solution, small anions may be essential
for Ih activation because the
HCO3
anions in the
Cl
-free solutions may probably contribute to
the Ih activation by small anions.
Physiological relevance of the hyperpolarization-activated inward current
Although a Na+ influx is involved in the
determination of the relatively positive RMP of Cajal-Retzius cells,
the RMP was not influenced by inhibitors of excitatory neurotransmitter
receptors or by the Na+ channel blocker TTX
(Mienville and Pesold 1999). These observations may
indicate the contribution of an inward current through
Ih channels to the RMP. However, our
observations that Ih activated at
potentials negative to
90 mV and that a block of
Ih by Cs+ was
without effect on the RMP strongly suggest that
Ih does not contribute to the
relatively positive RMP of Cajal-Retzius cells.
It was already shown that Ih
contributes to membrane oscillations by intervening with the
afterhyperpolarization (McCormick and Pape 1990a).
Because Cajal-Retzius cells express a strong afterhyperpolarization, we
also tested if a block of Ih by
Cs+ had an effect on the properties of action
potentials. However, we could not find an effect of
Cs+ on the afterhyperpolarization nor on other
action potential properties. This observation suggests that
Ih does probably not contribute to the
active membrane properties. Because we could not find an effect of
blocking Ih on any
electrophysiological properties, the physiological relevance of
Ih for Cajal-Retzius cells is
currently unknown.
One possible hypothesis for the role of
Ih may be that during development
either the activation threshold of Ih
is shifted to more positive potentials or that RMP is shifted to more
negative potentials. However, no developmental alteration in the
hyperpolarization-activated voltage sag had been found between P0 and
P6 (Zhou and Hablitz 1996a) and the RMP of Cajal-Retzius
cells remained also relatively positive during their development
(Mienville and Pesold 1999
). Another possibility is that
Ih could be activated by
hyperpolarizations, which occur during hypoxic events (Higashi
et al. 1988
; Luhmann 1996
; Luhmann and
Heinemann 1992
). And finally, it had been reported for a
variety of preparations that neurotransmitters can affect the
Ih by a shift in voltage dependence
(Colino and Halliwell 1993
; Li et al.
1993
; McCormick and Pape 1990a
; Pape and
McCormick 1989
). Because monoaminergic and serotonergic fibers
are among the first projections reaching the neocortex during early
development (Bayer and Altman 1991
), the
neurotransmitters released from these fibers may shift
Ih activation threshold to more
physiological Em. A functional
alteration of Ih due to noradrenaline
or other monoamines may thus be involved in the regulation of cortical development because it had been shown that noradrenaline determines the
fate of Cajal-Retzius cells (Naqui et al. 1999
).
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ACKNOWLEDGMENTS |
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The authors thank P. Schwarz and B. Hellmuth for helpful assistance.
This work was supported by Deutsche Forschungsgemeinschaft Grants Lu 375/3-2 and SFB 194/B4 to H. J. Luhmann.
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FOOTNOTES |
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Address for reprint requests: W. Kilb, Institut für Neurophysiologie, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, D-40001 Düsseldorf, Germany (E-mail: kilb{at}uni-duesseldorf.de).
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 3 February 2000; accepted in final form 25 April 2000.
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REFERENCES |
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