1Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724; and 2The National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224
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ABSTRACT |
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Lupica, Carl R.,
James A. Bell,
Alexander F. Hoffman, and
Patricia L. Watson.
Contribution of the Hyperpolarization-Activated Current
(Ih) to Membrane Potential and
GABA Release in Hippocampal Interneurons.
J. Neurophysiol. 86: 261-268, 2001.
Intrinsic GABAergic
interneurons provide inhibitory input to the principal neurons of the
hippocampus. The majority of interneurons located in stratum oriens
(s.o.) of the CA1 region express the hyperpolarization-activated cation
current known as Ih. In an effort to
elucidate the role of this current in regulating the baseline
excitability of these neurons and its participation in the regulation
of the release of GABA onto CA1 pyramidal neurons, we utilized whole
cell electrophysiological recordings from both populations of cells. In
voltage-clamp experiments, hyperpolarization of the interneuron
membrane initiated a large inward current with an estimated activation
threshold of 51.6 ± 7.6 mV and a half-maximal voltage of
73.0 ± 7.0 mV. This current was blocked by bath application of
the Ih inhibitors ZD 7288 (50 µM) or
cesium (2 mM). Current-clamp experiments at the interneuron resting
membrane potential (
61.3 ± 1.2 mV) revealed a significant
hyperpolarization, a decrease in the rate of spontaneous action
potential discharge, an increase in the cellular input resistance, and
the elimination of rebound afterdepolarizations during blockade of
Ih with ZD 7288 (50 µM). The
hyperpolarizing effect of ZD 7288 was also substantially larger in
interneurons clamped near
80 mV using current injection through the
pipette. In addition to neurons exhibiting
Ih, recordings were obtained from a
small population of s.o. interneurons that did not exhibit this
current. These cells demonstrated resting membrane potentials that were
significantly more negative (
73.6 ± 5.5 mV) than those observed
in neurons expressing Ih, suggesting that this current contributes to more depolarized membrane potentials in these cells. Recordings from postsynaptic pyramidal neurons demonstrated that blockade of Ih with
ZD 7288 caused a substantial reduction (~43%) in the frequency of
spontaneous action potential-dependent inhibitory postsynaptic currents
(IPSCs), without altering their average amplitude. However, miniature
action-potential-independent IPSC frequency, amplitude, and decay
kinetics were unaltered by ZD 7288. These data suggest that
Ih is active at the resting membrane potential in s.o. interneurons and as a result contributes to the
spontaneous activity of these cells and to the tonic inhibition of CA1
pyramidal neurons in the hippocampus.
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INTRODUCTION |
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Voltage-dependent ion
channels are ultimately responsible for changing the amount or pattern
of neurotransmitter released from neurons. However, the contributions
that these ion channels make to the process of neurotransmitter release
in most neuronal networks remains incompletely understood.
Hyperpolarization-activated cation channels that carry inwardly
rectifying currents (termed If) were
first identified in cardiac sinoatrial cells (Brown and DiFrancesco 1980; DiFrancesco 1981
) and were
subsequently characterized in many different neuronal populations in
the CNS (for review, see Pape 1996
). In neurons, this
inward cation current (known as Ih) is
carried by Na+ and K+,
activates slowly, does not inactivate during prolonged
hyperpolarization, and possesses a reversal potential (
30 to
50 mV)
that is positive to the neuronal resting membrane potential
(Halliwell and Adams 1982
; Maccaferri and McBain
1996
; Mayer and Westbrook 1983
; Svoboda and Lupica 1998
). Because of these biophysical properties,
these ion channels are proposed to contribute to the neuronal resting membrane potential (RMP), patterns of rhythmic action potential discharge, and may provide a mechanism whereby strong membrane hyperpolarizations are opposed (Maccaferri and McBain
1996
; Solomon and Nerbonne 1993
; Svoboda
and Lupica 1998
). Additionally, this current can
presynaptically facilitate neurotransmitter release at the crayfish
neuromuscular junction (Beaumont and Zucker 2000
) and
postsynaptically regulate the shape of synaptic potentials during the
process of synaptic integration in the mammalian CNS (Magee
1998
, 1999
).
Hyperpolarization-activated cation channels are represented by the
products of at least four genes [termed hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective, HCN1 to -4 (Biel et al. 1999; Gauss et al. 1998
;
Ludwig et al. 1998
; Santoro et al.
1998
)]. Furthermore the homomeric ion channels derived from the expression of these genes possess distinct biophysical properties and are differentially sensitive to modulation by cyclic
adenosine-3'-5'-monophosphate (cAMP) and the ion channel blocker cesium
(Ludwig et al. 1998
, 1999
; Santoro et al. 1998
,
2000
). Messenger RNAs for HCN1, -2, and -4 genes are most
prominently represented in the CNS where localization studies have
revealed a heterogeneous distribution. In particular, HCN1 and HCN2 are
expressed at high levels throughout several cortical areas and in the
hippocampal formation (Moosmang et al. 1999
;
Santoro et al. 2000
).
Whereas the function of Ih has been
well defined in thalamic relay neurons where it regulates neuronal
firing patterns (Bal and McCormick 1996; Luthi
and McCormick 1998
; McCormick and Pape 1990b
),
its role in controlling neuronal activity throughout most of the brain
is not well defined. In the hippocampus,
Ih has been identified in both
principal output neurons (pyramidal neurons) (Gasparini and
DiFrancesco 1997
; Santoro et al. 2000
) and in
local circuit GABAergic interneurons (Maccaferri and McBain
1996
; Santoro et al. 2000
; Svoboda and
Lupica 1998
). These GABAergic interneurons play an important
role in regulating hippocampal activity and output because although
comprising a relatively small fraction of the total hippocampal
neuronal population (~10%), they each form multiple synapses on
hundreds of pyramidal neurons (Freund and Buzsaki 1996
).
This divergent synaptic arrangement thereby permits these interneurons
to synchronize the activity of a large number of principal output
neurons and initiate neuronal network oscillations that appear to be
important for information processing (Cobb et al. 1995
;
Whittington et al. 1995
). We have previously demonstrated that 90% of the GABAergic interneurons with somata located in stratum oriens (s.o.) of the CA1 region of the hippocampus exhibited Ih currents, as defined both
biophysically and by sensitivity to the
Ih blockers cesium and ZD 7288 (Svoboda and Lupica 1998
). In this study, we also
demonstrated that Ih contributed as
much as 60% of the whole cell conductance on hyperpolarization of the interneuron membrane and that inhibition of
Ih by µ- and
-opioid receptors
could generate outward currents in neurons voltage clamped near the RMP
(Svoboda and Lupica 1998
). These data suggested that Ih may be active near the RMP of these
neurons and therefore may play a significant role in regulating s.o.
interneuron excitability, GABA release onto CA1 pyramidal neurons, and
ultimately hippocampal output. However, because in these previous
studies interneurons were clamped at potentials that were likely
negative to their actual RMPs (Maccaferri and McBain
1996
; Svoboda and Lupica 1998
), the contribution
of Ih to the actual interneuron RMP
could not be assessed. Therefore the goals of the present study were to determine whether Ih contributes to
the RMPs of these s.o. interneurons and to define the role that this
current plays in the regulation of GABA release onto pyramidal neurons
in the CA1 region of the hippocampus. A portion of these results has
appeared in a preliminary report (Lupica et al. 1999
).
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METHODS |
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Brain-slice preparation
Hippocampal slices were prepared and maintained as previously
described (Miller et al. 1997; Svoboda and Lupica
1998
). Briefly, 14- to 30-day-old male Sprague-Dawley rats
(Sasco, Omaha, NE or Charles River Labs, Raleigh, NC) were killed by
decapitation. Their brains were removed and placed in ice-cold,
oxygenated artificial cerebral spinal fluid (ACSF; see following text).
Brain slices containing the hippocampus were cut transverse to the
anterior-posterior axis at 300 µm nominal thickness using a vibrating
tissue slicer (Technical Products International, St. Louis, MO). The
slices were then suspended on netting in a beaker containing ACSF that was aerated continuously with 95% O2-5%
CO2, at room temperature. Control ACSF consisted
of (in mM) 126 NaCl, 3.0 KCl, 1.5 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 11.0 glucose, and
26 NaHCO3, and saturated with 95%
O2-5% CO2. After
60 min
of incubation in the ACSF, a single slice was transferred to a
recording chamber (~250 µl vol) that was built into the stage of an
upright microscope (Carl Zeiss Instruments, Oberkochen, Germany).
Interneuron recordings
Interneuron somata were visualized in the s.o. of area CA1 using
a fixed stage upright microscope equipped with differential interference contrast optics and an infrared light source (DIC-IR) as
previously described in detail (Dodt and Zieglgansberger
1990; Miller et al. 1997
; Svoboda and
Lupica 1998
; Svoboda et al. 1999
). Whole cell
recordings were obtained from interneurons at room temperature
(20-23°C) using an Axopatch-200A or Axoclamp-2A amplifier (Axon
Instruments, Burlingame, CA) and electrodes pulled from thick-walled
borosilicate capillary tubing (0.75 mm ID, 1.5 mm OD, Sutter
Instrument, Novato, CA). The electrodes had resistances of 4-7 M
,
when filled with (in mM) 125.0 K-gluconate, 10.0 KCl, 10.0 HEPES, 1.0 EGTA, 0.1 CaCl2, 2.0 Mg2+-ATP, and 0.2 Na+-GTP
(adjusted to pH 7.2-7.4 with 1 M KOH, 270-280 mOsm). All recordings
were corrected for an 11.5-mV liquid junction potential measured
according to the method of Neher (1992)
. Series
resistance was generally <15 M
and was monitored throughout the
experiments by measuring the capacitative currents generated by small
(
5 mV, 250 ms) voltage steps (or current steps during current-clamp recordings). Cells were rejected from analysis if the series resistance changed by 10-15%. Voltage-clamp protocols were delivered using a
pulse generator (AMPI Master 8, Jerusalem, Israel), and signals were
acquired using a National Instruments Lab PC 1200 A/D converter (Austin, TX) and the Strathclyde electrophysiology software package (courtesy of Dr. John Dempster, Strathclyde University, Glasgow, UK,
http://innovol.sibs.strath.ac.uk/physpharm).
Pyramidal neuron recordings
Action-potential-dependent spontaneous inhibitory postsynaptic
currents (sIPSCs), and action-potential-independent miniature IPSCs
(mIPSCs) were recorded in cells voltage clamped at 70 to
90 mV
using the Axopatch-200A amplifier and whole cell electrodes filled with
the following solution (in mM): 125.0 CsCl, 10.0 HEPES, 1.0 EGTA, 0.1 CaCl2, 2.0 Mg2+-ATP, and
0.2 Na+-GTP, and the quaternary lidocaine
derivative QX-314, 2 (pH 7.2-7.4). QX-314 was added to eliminate
Na+-dependent action potentials and to block
Ih channels in the pyramidal neurons
from which whole cell recordings were made (Perkins and Wong
1995
). sIPSCs and mIPSCs were also pharmacologically isolated from excitatory postsynaptic currents (EPSCs) by addition of the glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX,
10 µM) and D-(
)-2-amino-5-phosphonopentanoic acid (APV, 40 µM) to the ACSF. sIPSCs and mIPSCs were amplified 5- to 10-fold, filtered at 3-5 kHz, and 3 min epochs of data were acquired (at 4-10
kHz) directly onto the hard drive of a personal computer.
Chemicals
Drugs were obtained from the following sources: tetrodotoxin (TTX) and QX-314, Alomone Laboratories (Jerusalem, Israel); DNQX, APV, and CsCl, Sigma (St. Louis, MO); ZD 7288, Tocris Cookson (Ballwin, MO). All drugs and channel blocking agents were made at 100 times their final concentration in de-ionized water and added to the ACSF bathing the slice (flow rate = 2 ml/min) using calibrated syringe pumps (Razel Scientific Instruments, Stamford, CT).
Analysis
The frequency, amplitudes, and kinetic properties of s- and
mIPSCs were analyzed using the Mini Analysis software package (v4.3,
Synaptosoft, Leonia, NJ, http://www.synaptosoft.com). In addition,
averaged s- and mIPSCs were generated by aligning individual events by
rise time, and a peak to decay single exponential fit was applied to
each average using the formula y = A1*
exp(x/
) + Baseline, where A1 is
the peak amplitude and
is the time constant for decay.
In some cases, hyperpolarization-activated currents that were sensitive
to ZD 7288 were plotted against the step voltage and fitted using the
Boltzmann equation
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Group data are presented as the means ± SE in all cases. Drug-induced changes in cumulative s- and mIPSC amplitude and interevent interval distributions were analyzed for statistical significance using the Kolmogorov-Smirnov (K-S) test (Mini Analysis v4.3), and a conservative critical probability level of P < 0.01. All other statistical tests, including t-tests and ANOVAs, were performed using a critical probability of P < 0.05 (Prism version 3.0, GraphPad Software, San Diego, CA). Post hoc analyses (Newman-Keuls test) were performed only when an ANOVA yielded a significant (P < 0.05) main effect.
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RESULTS |
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Properties of Ih in hippocampal stratum oriens interneurons
As described previously, membrane hyperpolarization under
voltage-clamp conditions produced a slowly activating inward current in
the majority of interneurons possessing somata located in s.o. of the
CA1 region of the hippocampus (Maccaferri and McBain
1996; Svoboda and Lupica 1998
). In interneurons
voltage clamped at
40 mV, the inward current reached a maximum when
the membrane was stepped to approximately
90 to
110 mV, and its
amplitude did not decay during a 1.5-s voltage step (Fig.
1, A and C). This inward current was blocked by ZD 7288 (50 µM; Fig. 1), and cesium (2 mM, not shown); both of which have been shown to inhibit
Ih in these neurons (Maccaferri
and McBain 1996
; Svoboda and Lupica 1998
), and
the cardiac equivalent of this current
(If) in sinoatrial pacemaker cells
(BoSmith et al. 1993
). The effect of ZD 7288 was time
dependent, reaching a maximum in approximately 5-7 min, and was
relatively selective for Ih since the
steady-state (ss) current measured near the end of 1.5-s constant
voltage step was reduced to a much larger extent than the instantaneous
current (ins) measured near the beginning of the voltage step (Fig. 1,
C and D). Because of this relationship,
Ih amplitude was measured by
subtracting Iins from
Iss at each hyperpolarizing voltage
step (Fig. 1D) as previously described (Svoboda and
Lupica 1998
).
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Contribution of Ih to membrane potential in s.o. interneurons
The hyperpolarization-activated current was not routinely
detected at membrane potentials near the voltage-clamp holding
potential of 40 mV, suggesting that its activation threshold was at a
more negative value (Fig. 1A). In an effort to estimate the
activation threshold for Ih, the peak
amplitude of the ZD 7288-sensitive component of the inward current was
plotted against the membrane potential
(Vm) at various hyperpolarized voltage
steps. The linear portions of these curves (4-5 points between
70
and
90 mV) were then fit using linear regression, and the activation
thresholds estimated from the x intercept (Kilb and
Luhmann 2000
). Using this analysis, the activation threshold
for Ih was estimated to be
51.6 ± 7.6 mV (n = 5; Fig. 1B). Since the mean
RMP of another group of s.o. interneurons, under identical recording
conditions, was found to be
61.3 ± 1.2 mV (n = 13) and the reversal potential of Ih
in these cells is approximately
30 mV (Maccaferri and McBain 1996
; Svoboda and Lupica 1998
), it is likely
that Ih was active at rest and thereby
may have exerted a tonic depolarizing influence on these neurons. To
test this hypothesis, we recorded from s.o. interneurons at their RMP
(i.e., no holding current applied) under current-clamp conditions in
the presence of tetrodotoxin (TTX, 500 nM), to block spontaneous action
potential discharge, and applied ZD 7288 (50 µM). Application of ZD
7288 for 10 min caused a significant hyperpolarization (to
66.6 ± 1.4 mV, P < 0.01, paired t-test),
greatly increased the cellular input resistance measured at steady
state and eliminated the rebound action potential (i.e., the
afterdepolarization) (Luthi and McCormick 1998
;
Pape 1996
) commonly observed at the termination of the
current step (Fig. 2A). To
determine whether this hyperpolarization was sufficient to decrease the
rate of action potential discharge, we recorded from a separate group
of s.o. interneurons in the absence of TTX pretreatment. As reported in
previous studies (Maccaferri and McBain 1996
;
Svoboda and Lupica 1998
), the majority (i.e., 10 of 13 in the present study) of s.o. interneurons were spontaneously active.
Furthermore, when ZD 7288 (50 µM) was applied, the frequency of
action potential discharge was significantly reduced in every spontaneously active cell (n = 10; Fig.
3). These data suggest that
Ih contributes to baseline s.o.
interneuron excitability.
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In addition to contributing to the RMP, another of the proposed roles
of Ih is to offset strong membrane
hyperpolarizations and to return the membrane potential to values
closer to action potential threshold (Pape 1996;
Svoboda and Lupica 1998
). This is because the
hyperpolarization-activated inward current is larger at more negative
membrane potentials, thereby exerting a stronger depolarizing
influence. Because of this property blockade of
Ih by ZD 7288 should result in a
larger hyperpolarization of the membrane potential in cells that are
more strongly inhibited. To test this, a separate group of s.o.
interneurons was current clamped to approximately
80 mV, using
current injection through the pipette. Then ZD 7288 (50 µM) was
applied for 10 min while measuring the membrane potential and cellular
input resistance. In these neurons, ZD 7288 caused a much larger
hyperpolarization (
99.6 ± 2.9 mV, P < 0.001, ANOVA) than that seen in the cells recorded at RMP and again blocked
the afterdepolarization normally seen on termination of strong
hyperpolarizing current pulses (Fig. 2B). Blockade of
Ih by ZD 7288 also resulted in a
substantial increase in the input resistance that was not significantly
different from that measured in the neurons recorded at rest (Fig.
2B). These data suggest that
Ih is active near the RMP of s.o.
interneurons and that it exerts a strong repolarizing influence on
hyperpolarized neurons.
The preceding data indicated that tonic activation of
Ih contributes to setting the RMP of
s.o. interneurons at more depolarized levels. Therefore interneurons
lacking Ih should exhibit more hyperpolarized RMPs and should also be insensitive to the effects of ZD
7288. Approximately 10% of all interneurons with somata located in
s.o. do not exhibit the membrane sag on hyperpolarization that is
associated with Ih (Svoboda and
Lupica 1998). We recorded from a sample of these neurons
(n = 5) in the present study and found that their RMPs
were significantly hyperpolarized (
73.6 ± 5.5 mV) when compared
with the majority of s.o. interneurons that exhibited
Ih (
61.3 ± 1.2 mV,
n = 13, P < 0.01; Fig.
4). In addition, the interneurons lacking
Ih were completely insensitive to ZD
7288 and did not exhibit afterdepolarizations (Fig. 4).
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Contribution of Ih to spontaneous GABA release onto CA1 pyramidal neurons
A previous anatomical study in this laboratory demonstrated that a
portion of hippocampal interneurons with somata located in s.o.
possessed axons that heavily innervated CA1 pyramidal neuron somata
(Svoboda et al. 1999). This suggested that this population of cells might provide strong GABAergic inhibition of CA1
pyramidal neurons and thereby contribute to the tonic modulation of
their output. In an effort to determine whether
Ih contributes to the tonic inhibition
of CA1 pyramidal neurons in hippocampal slices, we measured
spontaneously occurring IPSCs in these cells under control conditions,
and during blockade of Ih by ZD 7288. sIPSCs represent the postsynaptic response to spontaneously released quanta of GABA acting at GABAA receptors (e.g.,
see Hoffman and Lupica 2000
; Lupica 1995
)
and are thereby thought to reflect the activity of presynaptic
GABAergic interneurons. In the absence of Na+
channel blockade with TTX (see following text), sIPSCs are composed of
both action-potential-dependent and -independent populations. Previous
studies have also shown that the frequency of sIPSCs can be modified by
neuromodulators that regulate the presynaptic RMP (e.g., see
Miller et al. 1997
). sIPSCs measured in CA1 pyramidal neurons voltage clamped at
70 to
90 mV using symmetrical
concentrations of Cl
, and in the presence of
ionotropic glutamate receptor antagonists (DNQX, 10 µM; APV, 40 µM), demonstrated baseline frequencies of 5.8 ± 0.6 Hz
(range = 3.1-13.0 Hz, n = 11), and baseline
amplitudes of
23.5 ± 1.0 pA (range =
11.9 to
37.4 pA,
n = 11). These values compare favorably with those
reported previously by this and other laboratories in the same
preparation (Cohen et al. 1992
; Hoffman and
Lupica 2000
; Lupica 1995
; Miller et al.
1997
). Addition of ZD 7288 (50 µM) to the superfusion medium
resulted in a significant decrease in sIPSC frequency, in every cell
tested (P < 0.001, K-S test), that became maximal
after ~7-10 min. The average frequency of sIPSCs measured 10 min
after beginning ZD 7288 superfusion was 3.3 ± 0.2 Hz (range = 1.2-8.4 Hz, n = 11), representing a decrease of
~43% (Fig. 5; P < 0.001, paired t-test). In contrast to the robust inhibition
of sIPSC frequency, blockade of Ih by ZD 7288 did not result in a significant change in mean sIPSC
amplitude (21.1 ± 0.7 pA, P > 0.05, n = 11, paired t-test; Fig. 5, B
and C), despite the observation that significant reductions
were observed in 2 of 11 neurons when the cumulative amplitude
distributions were analyzed using the K-S test (P < 0.001). Together, these data suggest that the blockade of
Ih in GABAergic interneurons by ZD
7288 resulted in a decrease in GABA release onto CA1 pyramidal neurons.
|
mIPSCs recorded during superfusion with TTX (500 nM) were also measured
in a different group of CA1 pyramidal neurons to determine whether
Ih plays a role in supporting quantal
neurotransmitter release in the mammalian CNS. Recording conditions
were similar to those used to measure sIPSCs except that TTX was added
to the superfusion medium for 15 min prior to application of ZD 7288. Previous studies in this laboratory have demonstrated that this protocol is sufficient to completely eliminate stimulation-induced neurotransmitter release and to completely block action potential generation in CA1 pyramidal neurons (Hoffman and Lupica
2000
; Lupica 1995
). Consistent with these
previous studies, TTX caused a large decrease in the frequency
(1.2 ± 0.2 Hz, range = 0.3-1.9 Hz, n = 15)
and amplitude (17.7 ± 0.5 pA, range = 10.8-25.3 pA, n = 15) of mIPSCs (Fig.
6). However, when mIPSCs were measured after 10 min of superfusion with ZD 7288, significant changes in
average frequency (1.3 ± 0.2 Hz, range = 0.2-2.2 Hz,
n = 15, P > 0.05) or amplitude
(18.8 ± 0.5 pA, range = 14.0-24.4 pA, n = 15, P > 0.05) were not observed (Fig. 6). Similarly ZD
7288 had no effect on the decay time constants of the mIPSCs
(control = 19.4 ± 0.2 ms, ZD 7288 = 19.8 ± 0.1 ms).
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DISCUSSION |
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The RMP of central neurons is established through
interactions among a variety of tonically active inward and outward
currents. The findings of the present study suggest that the inward
current, Ih, plays a significant role
in setting the RMP and baseline level of excitability of hippocampal
GABAergic interneurons found in the s.o. of area CA1. This conclusion
is supported by several pieces of data in the present study. First, the
estimated activation threshold of this voltage-dependent current (ca.
52 mV) and its reversal potential (ca.
30 mV) (Maccaferri
and McBain 1996
; Svoboda and Lupica 1998
) were
positive to the RMP of these cells (ca.
61 mV), suggesting that
Ih was partially active at rest.
Second, both cesium and the selective blocker of
Ih, ZD 7288 (BoSmith et al.
1993
), resulted in a significant hyperpolarization of the resting interneuron membrane potential that was consistent with the
blockade of this inward current. A final piece of data supporting the
idea that Ih contributes to a
significantly depolarized RMP was obtained from the small population of
s.o. interneurons that do not express this current (i.e., <10%)
(Svoboda and Lupica 1998
). These neurons exhibited RMPs
that were significantly more negative than those observed in the larger
population of s.o. interneurons in which
Ih was observed.
This supports the hypothesis that Ih contributes to more depolarized RMPs and suggests that the absence of
this current can serve as a predictor of the RMP. Collectively, these
data suggest that the inward Ih is
active at the RMP, thereby counterbalancing the tonically active
outward currents (i.e., K+ currents) that would
shift the membrane potential toward more hyperpolarized levels if
unopposed. At the present time, it is unclear why the membrane
potential did not more closely approach the K+
reversal potential (i.e., Ek =
96
mV) following blockade of Ih by ZD
7288. However, it is possible that additional inward currents are
present in these neurons that were not blocked by ZD 7288 or cesium.
The results of the present study also suggest that the depolarizing
influence of Ih is partly responsible
for the continued release of GABA onto postsynaptic CA1 pyramidal
neurons and thus the tonic inhibition of their activity. This is
supported by data demonstrating that application of ZD 7288 caused a
substantial decrease in the frequency of sIPSCs recorded in these
cells. Previous studies have provided strong evidence that the majority
of s- and mIPSCs recorded in hippocampal principal neurons arise from GABAergic interneurons, known as basket or perisomatic cells, that
provide dense synaptic input to their somata (Miles et al. 1996; Soltesz et al. 1995
). In the context of
the present study, this suggests that at least some of these neurons
express Ih channels that are active at
the RMP and that this current contributed to the continued release of
GABA and the tonic inhibition of CA1 pyramidal cells. We have
previously demonstrated that >90% of the interneurons with somata
located in s.o. of area CA1 express whole cell currents exhibiting
characteristics consistent with Ih
(Svoboda and Lupica 1998
). However, we also found that
only ~16% of these s.o. interneurons possessed axons that terminated in the CA1 pyramidal cell body layer (Svoboda et al.
1999
). Whereas it is possible that these neurons were primarily
responsible for the ZD 7288-sensitive sIPSCs observed in the present
study, it is also possible that other groups of interneurons with
somata located outside of s.o. that also express
Ih may project to s. pyramidale. In
fact, HCN mRNA expression (HCN1 and HCN2) that is likely associated
with hippocampal interneurons has been described in s.o., s.
pyramidale, s. radiatum, and s. lacunosum-moleculare in the CA1 region
of the hippocampus (Santoro et al. 1997
, 2000
). Thus
many different interneuron classes may express these
hyperpolarization-activated cation channels, and these cells may also
project to CA1 pyramidal neuron somata. Therefore we cannot exclude the
possible contribution that these neurons may have made to the ZD
7288-sensitive sIPSC population in the present study.
The absence of effects of ZD 7288 on action potential-independent
mIPSCs implies that the primary mechanism through which Ih alters tonic GABAergic inhibition
is by decreasing the somatodendritic excitability of hippocampal
interneurons. Furthermore, this suggests that
hyperpolarization-activated ion channels, which have been immunohistochemically localized to inhibitory basket cell terminals in
the hippocampus (Santoro et al. 1997), are probably not
more directly involved in the neurotransmitter release process under basal conditions. These data are in contrast to those observed at the
crayfish neuromuscular junction where
Ih facilitated action potential-independent neurotransmitter release (Beaumont and
Zucker 2000
), although this was observed only when the
magnitude of Ih was augmented via an
increase in cAMP accumulation (Beaumont and Zucker
2000
). In the present study, postsynaptic changes in GABA sensitivity or pyramidal neuron membrane time constants can be excluded
as possible explanations for the effect of ZD 7288 on sIPSCs since
mIPSC kinetics (i.e., decay time constants) were unaffected.
Furthermore, Ih was blocked in CA1
pyramidal neurons by the addition of QX-314 to the pipette solution
(Perkins and Wong 1995
), eliminating postsynaptic
blockade of Ih by ZD 7288 as an
explanation for the modulation of sIPSCs. Based on these observations
we hypothesize that blockade of Ih by
ZD 7288 resulted in the hyperpolarization of interneurons, a decrease
in action potential frequency, and a diminution of the amount of action potential-dependent GABA release onto the CA1 pyramidal neurons. In this respect these results are similar to those reported for cerebellar basket cells (Southan et al. 2000
). However,
it is not necessary to invoke the modulation of
Ih channels in the nerve terminals as
an explanation for the observed modulation by ZD 7288 because, as the
present data indicate, the reduction in somatic excitability appears to
be sufficient to explain the decrease in GABA release.
The hyperpolarization-activated current is modulated by
neurotransmitters in the peripheral and central nervous systems. This includes the facilitation of Ih by
monoamines such as norepinephrine and serotonin through a shift of the
voltage dependence of the current to more depolarized levels
(Bergles et al. 1996; Bobker and Williams
1989
; Ingram and Williams 1994
;
Maccaferri and McBain 1996
; McCormick and Pape
1990a
; Pape and McCormick 1989
). In addition, Ih is inhibited by µ-opioid
receptors in peripheral neurons (Ingram and Williams
1994
), by µ- and
-opioid receptors in central neurons (Svoboda and Lupica 1998
), and by adenosine in central
neurons (Rainnie et al. 1994
). Furthermore, adrenergic
receptor activation facilitates (Bergles et al. 1996
),
whereas opioid receptor activation inhibits spontaneous GABA release in
this preparation (Cohen et al. 1992
; Lupica
1995
). This has led to the hypothesis that the opioid-induced
inhibition and the adrenergic receptor-induced enhancement of
Ih would result in altered interneuron
excitability and corresponding changes in GABA release from these cells
(Svoboda and Lupica 1998
). However, because both
adrenergic and opioid receptors modulate additional ion channels (e.g.,
inwardly rectifying K+ channels) (Svoboda
and Lupica 1998
) in these same neurons, it has been difficult
to demonstrate this directly. Whereas the inhibitory effect of ZD 7288 on Ih is generally larger than that of
the opioids, qualitatively and functionally the effects on synaptic
transmission should be similar. Therefore the present results support
the hypothesis that neuromodulators that either positively or
negatively alter the contribution of
Ih to the interneuron RMP will in turn
predictably alter the amount of GABA released onto postsynaptic
principal neurons. Thus monoamines would be predicted to increase
(e.g., Bergles et al. 1996
) and opioids decrease
(Svoboda and Lupica 1998
) interneuron excitability and
GABA release via this mechanism. In this way the modulation of
Ih by neurotransmitter receptor activation and its altered contribution to the neuronal RMP may provide
an additional mechanism through which principal neuron activity and
output may be modified in the hippocampus and throughout the central
and peripheral nervous systems.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute on Drug Abuse Grant DA-07725.
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FOOTNOTES |
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Address for reprint requests: C. R. Lupica, Dept. of Pharmacology, Rm. 545, Life Sciences North, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724-5050 (E-mail: crlupica{at}u.arizona.edu).
Received 20 December 2000; accepted in final form 27 March 2001.
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REFERENCES |
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