Departments of Pediatrics and Neuroscience, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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Aoki, Takuya and
Scott C. Baraban.
Properties of a Calcium-Activated K+ Current on
Interneurons in the Developing Rat Hippocampus.
J. Neurophysiol. 83: 3453-3461, 2000.
Calcium-activated potassium currents have an essential role in
regulating excitability in a variety of neurons. Although it is well
established that mature CA1 pyramidal neurons possess a
Ca2+-activated K+ conductance
(IK(Ca)) with early and late components,
modulation by various endogenous neurotransmitters, and sensitivity to
K+ channel toxins, the properties of
IK(Ca) on hippocampal interneurons (or
immature CA1 pyramidal neurons) are relatively unknown. To address this
problem, whole-cell voltage-clamp recordings were made from visually
identified interneurons in stratum lacunosum-moleculare (L-M) and CA1
pyramidal cells in hippocampal slices from immature rats (P3-P25). A
biphasic calcium-activated K+ tail current was elicited
following a brief depolarization from the holding potential (50 mV).
Analysis of the kinetic properties of IK(Ca)
suggests that an early current component differs between these two cell
types. An early IK(Ca) with a large peak
current amplitude (200.8 ± 13.2 pA, mean ± SE), slow time
constant of decay (70.9 ± 3.3 ms), and relatively rapid time to
peak (within 15 ms) was observed on L-M interneurons
(n = 88), whereas an early IK(Ca) with a small peak current amplitude
(112.5 ± 7.3 pA), a fast time constant of decay (39.4 ± 1.6 ms), and a slower time-to-peak (within 26 ms) was observed on CA1
pyramidal neurons (n = 85). Removal of
extracellular calcium or addition of inorganic Ca2+ channel
blockers (cadmium, nickel, or cobalt) was used to demonstrate the
calcium dependence of these currents. Addition of norepinephrine, carbachol, and a variety of channel toxins (apamin, iberiotoxin, verruculogen, paxilline, penitrem A, and charybdotoxin) were used to
further distinguish between IK(Ca) on these
two hippocampal cell types. Verruculogen (100 nM), carbachol (100 µM), apamin (100 nM), TEA (1 mM), and iberiotoxin (50 nM)
significantly reduced early IK(Ca) on CA1
pyramidal neurons; early IK(Ca) on L-M
interneurons was inhibited by apamin and TEA. Combined with previous
work showing that the firing properties of hippocampal interneurons and
pyramidal cells differ, our kinetic and pharmacological data provide
strong support for the hypothesis that different types of
Ca2+-activated K+ current are present on these
two cell types.
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INTRODUCTION |
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Neuronal calcium-activated potassium current,
IK(Ca), has been extensively
characterized in both primary cultures and the in vitro slice
preparation (Meis and Pape 1997; Morita et al. 1982
; Sacchi et al. 1995
; Schwindt et al.
1992
). In hippocampal pyramidal cells, action potential firing
frequency, the rate of burst occurrence (Chamberlin and
Dingledine 1989
; Madison and Nicoll 1984
),
termination of epileptiform discharges (Alger and Nicoll
1980
), spike afterpolarization (Storm 1987
), and
afterhyperpolarizations following a spike train (Lancaster and
Adams 1986
) are regulated by a complex sequence of
Ca2+-activated K+
conductances. Three calcium-activated potassium current components have
been described for CA1 pyramidal neurons: a fast
IK(Ca) that activates rapidly (<5 ms)
after calcium influx, a medium IK(Ca) lasting 20-100 ms, and a slow IK(Ca)
that activates with a time constant >100 ms and decays over several
seconds (Lancaster and Adams 1986
; Lancaster and
Nicoll 1987
; Storm 1989
; Velumian and Carlen 1999
). In the adult hippocampus, a great deal of
information is already available concerning these
Ca2+-dependent K+ currents
e.g., second-messenger pathways, receptor systems, biophysical properties, and pharmacological sensitivities (Lancaster and
Nicoll 1987
; Madison and Nicoll 1982
, 1986a
,
1987
; Sah 1996
; Storm 1990
). For
example, charybdotoxin or high concentrations of tetraethylammonium chloride (TEA) reduce fast IK(Ca)
(Lancaster and Adams 1986
; Lancaster and Nicoll
1987
), whereas slow IK(Ca) is
abolished by neurotransmitters (e.g., acetylcholine, norepinephrine,
dopamine, and histamine) (Sah 1996
; Sah and
Isaacson 1995
) or iberiotoxin (Blatz and Magleby 1987
). At the same time, the properties of
IK(Ca) on developing CA1 neurons have
received relatively little attention (Costa et al. 1991
)
and virtually nothing is known about this current on hippocampal
interneurons.
Interneurons of stratum lacunosum-moleculare (L-M) of the CA1
subfield are predominantly GABAergic and exert extensive inhibitory control of pyramidal cells (Freund and Buzsaki 1996). It
is well established that the intrinsic firing properties of these
inhibitory hippocampal neurons are markedly different from responses of
excitatory pyramidal cells. For example, interneurons have
short-duration action potentials with prominent spike
afterhyperpolarizations (Kunkel et al. 1988
;
Lacaille and Schwartzkroin 1988
). Although the majority
of these cells are silent at resting membrane potential, depolarizing
current injection reveals a firing pattern characterized by
fast-spiking and little spike frequency adaptation (Williams et
al. 1994
). In contrast, CA1 pyramidal neurons are regularly spiking cells exhibiting a significant degree of spike frequency adaptation (Madison and Nicoll 1982
). In
voltage-clamp studies, it was determined that the macroscopic
properties of voltage-gated K+ channels differ
between pyramidal cells and interneurons. These differences between
pyramidal cells and interneurons are associated with differential
expression of Kv-type potassium channel subunits (Martina et al.
1998
; Massengill et al. 1997
), distinct
pharmacological sensitivities (Zhang and McBain
1995a
,b
), and unique physiological properties for the
voltage-gated K+ currents (Chikwendu and
McBain 1996
). Although evidence for a calcium-activated
K+ current on interneurons has been suggested by
current-clamp analysis (Lacaille and Schwartzkroin 1988
;
Williams et al. 1994
; Zhang and McBain
1995a
), direct characterization of this current using voltage-clamp techniques has not been performed.
In recent years, new electrophysiological techniques have been
developed that facilitate the study of identified neurons in an acute
slice preparation (Stuart et al. 1993). We utilized
these infrared video microscopy techniques (in combination with
voltage-clamp recordings) to examine the properties of
IK(Ca) on interneurons and pyramidal
cells in the developing hippocampus. In addition, novel K-Ca channel
toxins have recently become available (e.g., verruculogen, penitrem A,
and paxilline) (Knaus et al. 1994
; Sanchez and
McManus 1996
), and we used these toxins to further characterize IK(Ca). Our results indicate that
interneurons possess a class of calcium-activated
K+ current distinct from that found on
hippocampal pyramidal neurons. This current differs in both its kinetic
properties and pharmacological profile and may contribute to the
differences in firing patterns observed between these hippocampal cell types.
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METHODS |
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Hippocampal slice preparation
Transverse hippocampal slices (300 µm) were prepared from 3- to 25-day-old Sprague-Dawley rats. Briefly, rats were anesthetized and decapitated, and the brain was rapidly removed in ice-cold, oxygenated (95%O2-5%CO2) sucrose artificial cerebrospinal fluid (sACSF) consisting of (in mM) 220 sucrose, 3 KCl, 1.25 NaH2PO4, 1.2 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 dextrose (295-305 mOsm). The brain was then blocked and glued to the stage of a Vibroslicer (WPI) and hippocampal slices cut in 4° C oxygenated sACSF. The resulting slices were then transferred to a holding chamber where they were submerged in oxygenated normal ACSF (nACSF) consisting of (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 1.2 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 dextrose (295-305 mOsm). Slices were held at 37° C for 45 min and then at room temperature for 6-7 h; experiments were performed within 8 h of slice preparation. For each experiment, an individual slice was transferred to a submersion-type recording chamber (Warner Instrument), where it was continuously perfused with oxygenated nACSF at a rate of ~2.5 ml/min1 (33.2 ± 0.1° C, except as noted).
Whole cell recording
Tight-seal (4-16 G) whole cell voltage-clamp
recordings were made with an Axopatch 1-D amplifier (Axon Instruments)
with appropriate series and capacitance compensation (Hamill et
al. 1981
). Patch pipettes were pulled from 1.5-mm borosilicate
filament-containing glass tubing (WPI) using a two-stage process, fire
polished and coated with silicone elastomer (Sylgard, Dow-Corning).
Patch pipettes were filled with internal recording solution consisting
of (in mM) 135 K gluconate, 8 NaCl, 10 HEPES, 2 Na-ATP, and 0.2 Na-GTP (pH 7.4; 285-290 mOsm); solutions were filtered through a 2-µm filter (Millipore) prior to use. Extracellular recording solution consisted of nACSF supplemented with 0.5-1 µM tetrodotoxin. The pipette was positioned under visual control using a Zeiss Axioskop (Carl Zeiss, Germany) equipped with a water-immersion (×40) objective, differential interference contrast optics, and an infrared-sensitive camera (C2400, Hamamatsu, Japan). Cells were voltage-clamped at
50 mV
and depolarizing steps (25-400 ms) of sufficient amplitude (typically
+50 to +60 mV) to elicit a robust, unclamped Ca2+
action current was applied (Fig. 1)
(Pedarzani et al. 1998
; Sah and Isaacson
1995
; Zhang et al. 1995
). Using this protocol,
currents evoked during the command potential were not fully clamped
owing to space-clamp limitations. However, the voltage control during the IK(Ca) signal that develops after
the end of the depolarizing step can be well maintained
(Constanti and Sim 1987
; Zhang et al.
1995
). The magnitude (in pA) of
IK(Ca) was determined at the peak
(Fig. 1A). Time to peak (in milliseconds) was measured as the difference between peak current and offset of the depolarizing voltage step (Fig. 1A). Because a large capacitive artifact
often accompanies the voltage step offset, this measurement is regarded as an estimate of the actual time-to-peak current. The
time-dependent decay (
c of decay, in milliseconds) of
calcium-activated K+ currents was fitted between
90 and 10% of peak using a single exponential equation and a Chebyshev
fit (Fig. 1B); early and late current components were fitted
by separate time constants. Significant current rundown was observed
previously using a K+-gluconate-based patch
solution to record IK(Ca) in the
hippocampal slice preparation (Pedarzani et al. 1998
;
Zhang et al. 1994
). As these studies were performed at
room temperature, we initially tested the effects of temperature on
IK(Ca). Outward tail currents did not
rundown during the recording period and showed marked temperature
dependence (n = 12; Fig. 1C). As such, all
subsequent studies were performed at bath temperatures between 32 and
34°C. Voltage-clamp command potentials and post hoc current analysis was performed using pCLAMP software (Axon Instruments). Current records
were low-pass filtered at 2 kHz (
3 dB, 8-pole Bessel), digitized at
4-10 kHz using a Digidata 1200 A/D interface, and stored on a Pentium
II microcomputer (Dell Computer). Whole cell access resistance (6-23
M
) and holding current (0.00 to
0.2 pA) were continuously
monitored, and cells were discarded if either value changed by more
than 25% during the recording period (typically 20-45 min).
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Drugs
Tetraethylammonium chloride (TEA), artenerol (norepinephrine), carbachol, cadmium chloride, cobalt chloride, manganese chloride, nickel chloride, and all salts were purchased from Sigma (St. Louis, MO). Tetrodotoxin, paxilline, penitrem A, verruculogen, apamin, charybdotoxin, and iberiotoxin were purchased from Alomone Labs (Israel). Toxins were dissolved in dH2O, stored as frozen aliquots and thawed just prior to use. All drugs were dissolved in nACSF and applied to the slice via rapid bath perfusion.
Statistical analysis
To avoid errors associated with oversampling (or inadequate drug washout), only one cell was recorded per slice and each cell was exposed to only one drug challenge. Results are presented as means ± SE. Data was analyzed using a Student's t test on the SigmaStat program (Jandel Scientific). Significance level was taken as P < 0.01 except as noted.
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RESULTS |
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Kinetic properties of calcium-activated potassium currents in the developing hippocampus
Whole cell voltage-clamp recordings (n = 173) were
obtained from visualized interneurons in st. lacunosum-moleculare (L-M) or pyramidal neurons in CA1 (Fig. 2,
A and B) of the immature rat hippocampus
(P10-P20). A protocol involving short depolarizing voltage steps
(120-ms; Fig. 2A, inset) was used to elicit
depolarization-activated (presumably,
Ca2+-activated) outward tail currents. Increasing
the amplitude of the depolarizing commandthus increasing calcium
influx (Jahromi et al. 1999
; Lancaster and Adams
1986
)
enhanced outward tail current amplitude (Fig. 2,
A and B). The outward tail current on L-M
interneurons (n = 88) could be described as having two
distinct components: "early" and "late." The early component
had a relatively large peak current amplitude (200.8 ± 13.2 pA;
measurements were made following a 120-ms depolarizing step to 0 mV), a
slow decay
c (70.9 ± 3.3 ms), and an estimated time-to-peak of
14.2 ± 0.1 ms; the late current component exhibited a slow rising
phase and an estimated time to peak around 420 ms (Table
1). Similarly, outward tail current on
CA1 pyramidal neurons (n = 85) also had early and late
components. The early current component displayed a kinetic profile
distinct from that observed for the early current component on
interneurons e.g., rapid decay
c (39.4 ± 1.6 ms;
P < 0.001 Student's t test), small peak
amplitude (112.5 ± 7.3 pA; P < 0.001), and an
estimated time- to-peak of 25.5 ± 1.2 ms (P < 0.001). In contrast, the late current component had kinetic properties
that were not distinct from those measured for interneurons (Table 1).
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At a holding potential of 50 mV, brief (25-400 ms) depolarizing
voltage steps to 0 mV were used to further analyze and compare the
early current component (Fig.
3A). On both L-M interneurons and CA1 pyramidal neurons, current amplitude increased with longer depolarizing pulses. At each step duration tested, peak amplitude for
the tail current was greater for L-M interneurons in comparison with
CA1 pyramidal cells (Fig. 3B). In contrast, the estimated time-to-peak was always later for CA1 pyramidal neurons in comparison with L-M interneurons (Fig. 3C). Because reliable
measurements of the
c of decay could not be made during short
depolarizing steps (<50-ms), these data were not included. Taken
together, our results demonstrate that distinct types of
Ca2+-activated K+ current
are present on hippocampal interneurons and pyramidal cells.
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We next used brief depolarizing voltage steps (120-ms, 0 mV; 6 repetitions per cell; t 5 min) to study the kinetic
properties of IK(Ca) at various
postnatal ages (P3-P25). A distinct early IK(Ca) was observed on pyramidal cells
and interneurons at even the youngest ages studied (P3). The peak
amplitude for this current significantly increased during development
for both cell types (Fig. 4, A
and B). Although the decay
c for early
IK(Ca) on CA1 pyramidal cells was
shorter in younger animals and increased slightly with age (Fig.
4C), no developmental change was observed for early IK(Ca) on L-M interneurons (Fig.
4D).
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Calcium dependence of calcium-activated potassium currents in the developing hippocampus
To examine the calcium dependence of outward tail currents, we
used a variety of manipulations designed to block
Ca2+ entry into the cell. First, slices were
bathed in a nominally Ca2+-free artificial
cerebrospinal fluid solution (0 Ca2+/2 mM EGTA).
In whole-cell recordings from CA1 pyramidal neurons and L-M
interneurons, peak current amplitude was significantly reduced during
bath perfusion with 0 Ca2+/2 mM EGTA solutions
(Fig. 5, A and B).
An example of the effect of 0 Ca2+/2 mM EGTA
nACSF on early IK(Ca) is illustrated
in Fig. 5A1. Zero calcium solutions also abolished late
IK(Ca) observed on pyramidal cells
(n = 3) or interneurons (n = 2).
Second, slices were bathed in nACSF in which Ca2+
was replaced with saturating concentrations of inorganic calcium channel blockers (e.g., 200 µM cadmium, 2 mM cobalt, or 2 mM nickel). An example of the effect of 2 mM Co2+ nACSF on
early IK(Ca) is illustrated in Fig.
5B1. Inorganic Ca2+ channel blockers,
markedly reduced peak current amplitude on both CA1 pyramidal neurons
and L-M interneurons (Fig. 5, A2 and B2). The
late current component was completely abolished by these manipulations
(data not shown). Third, slices were bathed in nACSF supplemented with
the dihyropyridine L-type Ca2+-channel blocker,
nifedipine (Fox et al. 1987). Nifedipine (5 µM)
reduced early IK(Ca) on L-M
interneurons but had little effect on early
IK(Ca) on CA1 pyramidal cells (compare
Fig. 5, A2 and B2). These results suggest that
L-type Ca2+ channels participate in the
generation of interneuron, but not pyramidal cell, early
IK(Ca). Collectively, our data suggest
that Ca2+ entry is responsible, at least in part,
for generation of IK(Ca) in the
immature rat hippocampus.
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Calcium-activated potassium currents of immature hippocampal neurons
RESPONSE TO NEUROTRANSMITTERS.
The hippocampus receives a rich innervation by catecholaminergic and
cholinergic fibers (Lewis et al. 1967; Loy et al.
1980
), and it is well established that these systems modulate
the firing activity of hippocampal neurons in adult hippocampus
(Madison and Nicoll 1982
, 1986
; Madison et al.
1987
). To further characterize IK(Ca) in the immature hippocampus, we
tested the effects of norepinephrine and carbachol on
IK(Ca). Early
IK(Ca) on CA1 pyramidal cells was markedly inhibited by bath application of 100 µM carbachol, whereas 10 µM norepinephrine had little effect on this current (Fig.
6A). An example of the effect
of 100 µM carbachol on IK(Ca) is
illustrated in Fig. 6A. In contrast, early
IK(Ca) on L-M interneurons was not
altered during bath perfusion with either norepinephrine or carbachol
(Fig. 6A2). NE and carbachol abolished late
IK(Ca) observed on pyramidal cells
(n = 5; Fig. 6, B and C) or
interneurons (n = 2) as expected (Lancaster and
Nicoll 1987
; Sah and Isaacson 1995
).
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PHARMACOLOGY.
To obtain a pharmacological profile for early
IK(Ca) on both L-M interneurons and
CA1 pyramidal cells, we tested a combination of established and
putative Ca2+-activated K+
channel blockers. Apamin and tetraethylammonium chloride (TEA) have
been used to block IK(Ca) on mature
hippocampal neurons (Lancaster and Nicoll 1987;
Stocker et al. 1999
; Williamson and Alger
1990
). Bath application of 100 nM apamin produced a modest
(20-30%) inhibition of early IK(Ca)
on immature CA1 pyramidal cells and a profound (more than 75%)
inhibition of early IK(Ca) on L-M
interneurons (Fig. 7, A2 and
B2). Bath application of a high concentration of TEA (1 mM)
inhibited the early current component on both cell types (Fig. 7,
A2 and B2). The effects of both drugs were
reversible on washout (20-40 min). A significant inhibition of early
IK(Ca) on CA1 pyramidal cells was also
observed during bath application of 100 nM verruculogen (Fig. 7A,
1 and 2). However, the same concentration of
verruculogen did not significantly reduce early
IK(Ca) on L-M interneurons (Fig.
7B, 1 and 2). 50 nM iberiotoxin was similarly effective at reducing early IK(Ca) on
CA1 pyramidal cells but not early
IK(Ca) on L-M interneurons. Bath
application of nACSF containing 100 nM paxilline, 100 nM penitrem A, or
100 nM charybdotoxin did not significantly inhibit
IK(Ca) (Fig. 7, A2 and
B2). Taken together, these results indicate that potassium
channel activity is responsible, at least in part, for generation of
IK(Ca).
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DISCUSSION |
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Previous studies (Lancaster and Nicoll 1987;
Sah 1996
; Storm 1989
, 1990
) indicated
that calcium-activated K+ current on mature
hippocampal CA1 pyramidal neurons is made up of at least two distinct
components: early (e.g., "fast" and "medium") and late (i.e.,
"slow"). Using whole-cell voltage-clamp analysis in immature tissue
slices, we extend these findings and show (for the first time) that
calcium-activated K+ current on hippocampal
interneurons is also made up of at least two distinct components. The
late, or slow, current component had similar kinetic and
pharmacological properties for both cell types. In both L-M
interneurons and CA1 pyramidal neurons, this current peaked between 400 and 500 ms, was abolished by application of inorganic
Ca2+ channel blockers, and was inhibited by
acetylcholine and norepinephrine. Sah and Clements
(1999)
reported a decay time constant of 1.5 ± 0.2 s for the slow calcium-activated K+
current on hippocampal CA1 pyramidal neurons
a value similar to that
reported here (see Table 1). The slow kinetics of this current may be
due to mobilization of Ca2+ from intracellular
stores (Sah and McLachlan 1991
), diffusion of
Ca2+ to K-Ca channels from remote sites of entry
(Lancaster and Zucker 1994
; Zhang et al.
1995
), or to delayed facilitation of L-type voltage-activated
Ca2+ channels (Cloues et al.
1997
). In contrast, the early current components displayed
distinct neuron-specific kinetic and pharmacological properties.
Several lines of evidence suggest that the early component we recorded
on immature CA1 pyramidal neurons is distinct from a fast, transient
Ca2+-dependent K+ current
previously identified on mature CA1 neurons (Lancaster and Adams
1986; Lancaster and Nicoll 1987
). First, fast
IK(Ca) (or fast AHP) activates in less
than 5 ms after calcium influx and decays with a time constant of
several hundred milliseconds; early
IK(Ca) activated more slowly and
decayed more rapidly. Second, fast
IK(Ca) is potently inhibited by low
concentrations of TEA (50-200 µM) or charybdotoxin (25 nM)
(Storm 1990
); high concentrations of TEA (1 mM) modestly
inhibited early IK(Ca), and 100 nM
charybdotoxin had no measurable effect on this current. Third,
consistent with a recent report (Stocker et al. 1999
),
we found an apamin-sensitive component of the early
IK(Ca) recorded on immature CA1
pyramidal neurons; apamin sensitivity has not been demonstrated for
fast IK(Ca) on mature hippocampal
pyramidal cells. One explanation for these differences is that fast
IK(Ca)
which has only been identified
in adult hippocampus (Lancaster and Adams 1986
;
Lancaster and Nicoll 1987
)
is a component of mature CA1
pyramidal neurons and was therefore not present in immature tissue
examined in our studies.
The early current component we recorded on CA1 pyramidal neurons is
probably analogous to previously identified medium
IK(Ca) (or medium AHP) on mature
hippocampal CA1 pyramidal neurons (Alger and Nicoll
1980; Storm 1989
; Williamson and
Alger 1990
). First, the kinetic properties of this current are
in good agreement with those reported for mature CA1 pyramidal neurons,
e.g., activates in ~25 ms following calcium influx and decays with a
time constant around 39 ms (Alger et al. 1994
;
Storm 1989
; Williamson and Alger 1990
).
Second, the pharmacological profile of neonatal early
IK(Ca) is similar to that reported for
medium AHP on mature CA1 pyramidal neurons (Lancaster and Nicoll
1987
; Williamson and Alger 1990
). Both currents
are significantly reduced by carbachol but largely insensitive to
norepinephrine. Third, estimated time-to-peak for early
IK(Ca) current (range: 8-58 ms)
closely parallels time-to-peak for the rise in free intracellular
calcium (range: 2-50 ms) measured in CA1 pyramidal neurons using
calcium fluorescence imaging (Sah and Clements 1999
).
Finally, an interesting characteristic of early
IK(Ca) on CA1 pyramidal neurons is
that it is potently inhibited by verruculogen
a novel indole alkaloid
previously shown to inhibit K-Ca channels on myocytes (Knaus et
al. 1994
). Because the functional role for early
IK(Ca) appears to be termination of
epileptiform burst discharges (Alger et al. 1980
, 1994
;
Storm 1989
; Williamson and Alger 1990
),
it is interesting to speculate that the "immature" properties of
early IK(Ca) in young animals
contributes to the enhanced seizure susceptibility observed at these
ages (Moshe et al. 1983
).
Here we have also characterized two components of
Ca2+-activated K+ current
present on st. lacunosum-moleculare interneurons of the immature
hippocampus. Previous studies from the McBain laboratory have shown
that st. oriens-alveus interneurons (Zhang and McBain 1995a,b
), parvalbumin-containing interneurons of st. pyramidale (Du et al. 1996
), and st. L-M interneurons
(Chikwendu and McBain 1996
) posses distinct repertoires
of voltage-gated K+ currents. These currents are
thought to play a role in defining the unique fast-spiking firing
pattern characteristic of interneurons (Kunkel et al.
1988
; Lacaille and Schwartzkroin 1988
;
Martina et al. 1998
; Storm 1990
). Our
data demonstrate that st. lacunosum-moleculare interneurons also
express an early calcium-dependent K+ current
distinct from that observed on CA1 pyramidal cells.
IK(Ca) in L-M interneurons is
dominated by a large, slowly decaying current with rapid onset. This
early current component was blocked by apamin and TEA, but largely
insensitive to carbachol, verruculogen, and iberiotoxin (i.e., drugs
which inhibit early IK(Ca) on CA1 pyramidal cells). A functional role for this early current component has not yet been identified, though it is likely that the unique neuron-specific properties of this current contribute to defining the
intrinsic firing properties of interneurons. Further, our data on the
kinetic and pharmacological properties of
IK(Ca) should be of considerable use
in establishing reliable models of interneuron physiology and lead to
direct testing of the roles played by various K+
currents in shaping firing activities.
In conclusion, despite the heterogeneity of hippocampal interneurons,
it is becoming clear that they possess distinct potassium channel
subunits. For example, fast-spiking interneurons in the dentate gyrus
express Kv3.1 and Kv3.2 mRNA for a voltage-activated K+ current that is highly sensitive to TEA and
4-aminopyridine (Martina et al. 1998) and interneurons
in st. oriens-alveus possess a K+ current with
sensitivity to apamin and insensitivity to IbTX (Zhang and
McBain 1995b
). Our voltage-clamp data support the
conclusion that distinct forms of ion channels are present on
inhibitory interneurons and provide direct evidence that an early
K+ current component on immature hippocampal
interneurons is a Ca2+-dependent current with
unique properties. A likely explanation for the differences between
early IK(Ca) on interneurons and
pyramidal cells is that K-Ca channel subunit expression is
neuron-specific. From molecular studies, we know that K-Ca channel
subtypes are prominently expressed in the hippocampal formation. In
particular, large-conductance BK (mslo), small-conductance
SK (rsk2 and hsk1), and intermediate-conductance
IK (Slack) channels are found in pyramidal and granule cell
layers of hippocampus (Joiner et al. 1998
; Knaus
et al. 1996
; Kohler et al. 1996
). However, it is
not clear at this time which K-Ca channel subunits are expressed by hippocampal interneurons. An alternative explanation for the
differences between early IK(Ca) on
interneurons and pyramidal cells could be that mechanisms to buffer
intracellular calcium or permit calcium entry into the cell via
membrane-bound channels are different between these two cell types.
Nonetheless we anticipate that interneurons, which represent an
important population of inhibitory neurons in the hippocampus
(Freund and Buzsaki 1996
), express a unique group of
K-Ca channel subunits and that our characterization of IK(Ca) will aide in the eventual
identification of these subunits as well as lead to a greater
understanding of the functional role of
Ca2+-activated K+ channels
in the CNS.
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ACKNOWLEDGMENTS |
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The authors thank R. A. Nicoll and H. L. Fields for critical comments on an earlier version of this manuscript. We also thank P. Castro for expert technical assistance.
This project was sponsored by a Junior Investigator grant from the Epilepsy Foundation of America (S. C. Baraban).
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FOOTNOTES |
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Present address and address for reprint requests: S. C. Baraban, Dept. of Neurological Surgery, Box 0520, University of California, San Francisco, 513 Parnassus Ave., San Francisco, CA 94143.
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 15 December 1999; accepted in final form 17 February 2000.
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