Centre de Recherche en Sciences Neurologiques et Département de Physiologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada
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ABSTRACT |
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Chapman, C. Andrew and Jean-Claude Lacaille. Intrinsic theta-frequency membrane potential oscillations in hippocampal CA1 interneurons of stratum lacunosum-moleculare. The ionic conductances underlying membrane potential oscillations of hippocampal CA1 interneurons located near the border between stratum lacunosum-moleculare and stratum radiatum (LM) were investigated using whole cell current-clamp recordings in rat hippocampal slices. At 22°C, when LM cells were depolarized near spike threshold by current injection, 91% of cells displayed 2-5 Hz oscillations in membrane potential, which caused rhythmic firing. At 32°C, mean oscillation frequency increased to 7.1 Hz. Oscillations were voltage dependent and were eliminated by hyperpolarizing cells 6-10 mV below spike threshold. Blockade of ionotropic glutamate and GABA synaptic transmission did not affect oscillations, indicating that they were not synaptically driven. Oscillations were eliminated by tetrodotoxin, suggesting that Na+ currents generate the depolarizing phase of oscillations. Oscillations were not affected by blocking Ca2+ currents with Cd2+ or Ca2+-free ACSF or by blocking the hyperpolarization-activated current (Ih) with Cs+. Both Ba2+ and a low concentration of 4-aminopyridine (4-AP) reduced oscillations but TEA did not. Theta-frequency oscillations were much less common in interneurons located in stratum oriens. Intrinsic membrane potential oscillations in LM cells of the CA1 region thus involve an interplay between inward Na+ currents and outward K+ currents sensitive to Ba2+ and 4-AP. These oscillations may participate in rhythmic inhibition and synchronization of pyramidal neurons during theta activity in vivo.
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INTRODUCTION |
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Rhythmic synchronization of principal neurons is
thought to contribute to mnemonic and information-processing
capabilities of the hippocampal formation. Rhythmic synchronization may
enhance cooperativity among efferents and transmission through
polysynaptic pathways (Jones 1993; Yeckel and
Berger 1990
) and may promote potentiation or depression of
synaptic inputs by generating alternate states of postsynaptic
depolarization and hyperpolarization (Chapman and Becker
1995
; Chapman and Racine 1997
; Huerta and
Lisman 1995
; Larson et al. 1986
; Pavlides
et al. 1988
; Singer 1993
). Both extrinsic inputs
(Petsche et al. 1962
) and intrinsic conductances of
principal neurons (Garcia-Munoz et al. 1993
;
Leung and Yim 1991
; Nuñez et al.
1987
) contribute to hippocampal theta-frequency (4-12 Hz) activity (Bland and Colom 1993
). The extrinsic input
that drives both hippocampal and entorhinal cortex theta rhythms in
vivo is derived from the medial septum (Alonso and Garcia-Austt
1987
; Mitchell et al. 1982
; Petsche et
al. 1962
), and theta-like activity also can be induced in vitro
by cholinergic agonism (Konopacki et al. 1987
;
MacVicar and Tse 1989
).
Hippocampal interneurons powerfully inhibit large numbers of pyramidal
neurons (Freund and Buzsáki 1996; Lacaille
et al. 1987
) and contribute to theta activity by rhythmically
inhibiting pyramidal cells (Fox 1989
; Leung
1984
; Tóth et al. 1997
; Ylinen et
al. 1995
). Each theta cycle is associated with activation of GABAA chloride conductances proximal to the soma of CA1
pyramidal cells (Buzsáki et al. 1986
;
Brankack et al. 1993
; Fox et al. 1983
;
Leung and Yim 1986
; Soltesz and Deschênes
1993
; Ylinen et al. 1995
). Further,
theta-frequency membrane potential oscillations in pyramidal cells in
vitro can be synchronized by rhythmic stimulation of basket cells
(Cobb et al. 1995
). Interneurons also may mediate much
of the septal contribution to hippocampal theta because septal cholinergic inputs contact both interneurons and pyramidal cells (Léránth and Frotscher 1987
) and GABAergic
inputs target predominantly interneurons (Freund and Antal
1988
; Gulyás et al. 1990
).
Although the physiological and morphological diversity of hippocampal
interneurons suggests that subtypes differ in their computational
roles, it is not known which interneuronal subtype(s) contribute most
critically to theta activity. Basket and axo-axonic cells can pace
theta-frequency membrane potential oscillations in pyramidal cells
(Cobb et al. 1995), but the high-frequency of
spontaneous synaptic activity and firing in these interneurons (Schwartzkroin and Mathers 1978
) suggest that they in
turn must be paced by other neurons. Interneurons located near the
border of stratum radiatum and stratum lacunosum-moleculare (LM)
display much less spontaneous synaptic activity, fire at slower rates, and show intrinsic membrane potential oscillations (Lacaille and Schwartzkroin 1988
; Williams et al. 1994
).
Therefore in addition to modulating the activity of other interneuron
subtypes (Hajos and Mody 1997
; Vida et al.
1998
), LM cells may provide rhythmic inhibition of CA1
pyramidal neurons and may mediate some of the influence of the septum
and entorhinal cortex on hippocampal theta activity (Freund and
Antal 1988
; Kunkel et al. 1988
; Witter et al. 1988
).
In the present experiments, we have characterized membrane potential oscillations in LM interneurons using whole cell current-clamp recordings in slices and have found that oscillations were generated by Na+ and K+ conductances. Using biocytin cell labeling we found that oscillations were expressed preferentially in interneurons in LM and occurred less often in interneurons located in stratum oriens.
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METHODS |
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Methods were similar to those reported previously
(Williams et al. 1994), and all chemicals were obtained
from Sigma (St. Louis, MO) unless otherwise indicated.
Hippocampal slices
Hippocampal slices were obtained from 4- to 6-wk-old Sprague Dawley rats (Charles River, Montréal) anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ) and decapitated. The brain was removed quickly from the skull and placed in cold (4°C) artificial cerebrospinal fluid (ACSF) that contained (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2.4 CaCl2, 26 NaHCO3, and 10 dextrose saturated with 95% O2-5% CO2. Transverse hippocampal slices (300-µm thick) were cut in cold oxygenated ACSF using a vibratome and then kept at room temperature.
After 1 h, slices were held submerged in a recording chamber and
viewed with an upright microscope (Carl Zeiss Axioskop, Jena, Germany)
equipped with Hoffman optics (Modulation Optics, Greenvale NY), a
long-range water immersion objective (×40), and an infrared video
camera (Cohu 6500, San Diego, CA). The chamber was perfused with
oxygenated ACSF at room temperature (22°C) at a rate of 3 ml/min.
Temperature was increased to 32 ± 1°C in experiments with five LM interneurons to monitor oscillations at more physiological temperatures (Dagan Corp. TC-10, Minneapolis, MN).
Whole cell recording
Patch pipettes for whole cell current-clamp recordings were
pulled from borosilicate glass (1.0 mm OD, 4-7 M) using a
horizontal puller (Sutter Instruments, P87, Novato, CA). Patch pipettes
were filled with a solution containing (in mM) 140 K-gluconate, 5 NaCl, 2 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 0.5 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 ATP-Tris, 0.4 GTP-Tris, and 0.1% biocytin (pH adjusted to 7.2-7.3 with KOH). Patch pipettes were placed in contact with soma
of interneurons under visual guidance and using gentle positive pressure. Tight seals (2-12 G
) were obtained under voltage clamp with the aid of gentle suction, and stronger suction was used to obtain
whole cell configuration. Current-clamp recordings were begun after a
5-min period to allow the patch solution and cell interior to
equilibrate. Membrane potential recordings (DC-3 kHz) were obtained
with an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA),
displayed on a digital oscilloscope (Gould 1604, Ilford, UK), and
digitized for storage on video cassette (Neuro Data Instruments,
NeuroCorder DR-886, New York, NY). Recordings also were analogue
filtered at 2 kHz (8-pole Bessel, Frequency Devices 900, Haverville,
MA), and digitized at 10 kHz (Axon, TL-1) for storage on computer hard
disk. Recordings were accepted if the series resistance was <50 M
(mean = 34 ± 1.3 M
) and if input resistance and mean
resting membrane potential were stable. Series resistance was monitored
repeatedly during each experiment.
The voltage dependence of membrane potential oscillations and their effect on cell discharge patterns were assessed by varying membrane potential relative to spike threshold with steady current injection. Cell discharge was monitored at spike threshold and at slightly more depolarized levels. Recordings were repeated at the same potentials after application of pharmacological agents, and membrane conductance blocks were assessed by monitoring changes in action potential and voltage response (at rest) to positive and negative current pulses (500-ms duration).
Pharmacology
Stock solutions were stored frozen and diluted in ACSF on the
day of experiments. Ionotropic glutamate and GABAA synaptic transmission were blocked with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM), (±)-2-amino-5-phosphonopentanoic acid (AP-5, 50 µM;
Research Biochemicals International, Natick, MA) and bicuculline methiodide (25 µM). Sodium currents were blocked with tetrodotoxin (TTX, 0.5 µM). The hyperpolarization-activated cationic current Ih (Hotson et al. 1979;
Maccaferri and McBain 1996a
) was blocked with CsCl (1 mM). Calcium- and Ca2+-dependent currents were blocked by
perfusing slices with Ca2+-free ACSF in which
Ca2+ was replaced by Mg2+ or by applying
CdCl2 (50 µM).
The K+ channel blockers Ba2+ (1 mM), 4-aminopyridine (4-AP, 50 µM or 10 mM), and tetraethylammonium (TEA, 30 mM) were applied in the presence of CNQX (20 µM), AP-5 (50 µM) and bicuculline (25 µM). A low concentration of TEA (100 µM) was applied in the absence of these antagonists. When millimolar concentrations of 4-AP and TEA were used, the GABAB receptor blocker CGP 55845A (1.0 µM; Ciba Geigy, Basel, Switzerland) also was added, and the ACSF osmolarity was held constant (300-310 mOsm) by reducing Na+ and replacing it in control ACSF with choline (ICN Biomedicals, Aurora, OH). When Ba2+ and Cd2+ were used PO4 and SO4 were removed.
Analysis
Samples of membrane potential (15-s duration) were prepared for routine spectral analysis by low-pass filtering at 40 Hz and reducing the effective sampling rate to 1 kHz. Average power spectra were calculated as the square of the magnitude of the FFT using the software package Origin (Microcal, Northampton, MA) based on three 2.048-s duration segments selected to contain no action potentials. The low-pass filter was set to 100 Hz instead of 40 Hz for preliminary assessment of oscillations in LM cells and for analysis of oscillations in interneurons in stratum oriens. Cells without a clear peak in the power spectrum were considered to be non oscillatory. Changes in peak frequency and total power between 2.0 and 5.4 Hz after pharmacological manipulations were assessed using matched samples t-tests or repeated measures ANOVAs when appropriate.
Electrophysiological properties of LM cells were analyzed using the
software package pClamp 6.0 (Axon). Action potential height was
measured from resting membrane potential and action potential duration
was measured at the base. Amplitude of afterhyperpolarizations was
measured relative to the base of action potentials. Input resistance
was determined from the peak voltage response to 100-pA current
pulses (500-ms duration). Inward rectification was quantified by
expressing peak input resistance as a proportion of the steady-state resistance measured at the end of the current pulse (rectification ratio). Membrane time constant was measured by fitting an exponential function to the transient voltage response evoked by small
hyperpolarizing current pulses that did not evoke
hyperpolarization-activated rectification. Data were expressed as
means ± SE.
Histology
Slices were fixed between two filter papers in 4% paraformaldehyde in 0.1 M phosphate buffer for 2-4 h. Slices then were rinsed in 0.1 M phosphate buffer and kept at 4°C. Slices were embedded in 1% agarose and cut on a vibratome in 60 µm sections. Sections were treated with 1% H2O2 for 30 min to eliminate endogenous peroxidases and then washed (4 × 5 min) in 2.5% dimethyl sulfoxide and 0.1% Triton X in 0.1 M phosphate buffer. Sections then were incubated in avidin-biotin complex (ABC kit, Vectors Lab, Burlingame, CA; dilution 1:200) for 24 h. After rinsing in 0.05 M Tris buffer, sections were incubated in a solution of 0.05% 3'3-diaminobenzidine 4 HCl, 0.02% NiS04, 0.1 M imidazole, and 0.001% H2O2 in Tris-buffered saline (0.9% NaCl). Sections then were rinsed in Tris-buffered saline and cleared in xylene. Axonal and dendritic arborizations of well-filled cells were traced with a camera lucida.
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RESULTS |
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Stable recordings were obtained from 92 cells in LM and 8 cells in
stratum oriens. The passive electrical properties and firing patterns
of LM cells were similar to those recorded in previous whole cell
studies (Khazipov et al. 1995; Williams et al.
1994
). They had a mean resting membrane potential of
56.5 ± 0.5 mV, a high-input resistance (280 ± 10 M
),
and a long membrane time constant (
m = 25.7 ± 1.3 ms). Action potentials (amplitude, 92.0 ± 1.3 mV; duration,
2.7 ± 0.1 ms) typically were followed by biphasic fast (fAHP;
amplitude, 9.0 ± 0.4 mV) and medium duration (mAHP; amplitude,
8.7 ± 0.4 mV) afterhyperpolarizations.
Hyperpolarization-activated inward rectification was observed in 51 of
92 cells (e.g., Fig. 6B1) and mean rectification ratio among
these cells was 1.33 ± 0.03. The low incidence of spontaneous
synaptic potentials in LM cells (Lacaille and Schwartzkroin
1988
) allowed membrane potential oscillations to be studied
routinely in the absence of glutamate and GABA antagonists.
Intrinsic voltage-dependent oscillations
LM cells usually did not fire action potentials at rest. When cells were depolarized near spike threshold by positive current injection (usually <50 pA), almost all LM cells (84 of 92 cells) demonstrated oscillations in membrane potential at a frequency of 2-5 Hz reflected by clear peaks in the power spectrum (Fig. 1). When oscillations reached suprathreshold voltage levels, they caused firing which varied from single spikes to clusters of two to five spikes evoked at the frequency of oscillations (e.g., Fig. 3). Oscillations were sensitive to membrane potential and were eliminated by hyperpolarizing cells 6-10 mV below spike threshold (Fig. 1, A and B). Oscillation frequency increased from 2.3 ± 0.1 Hz to 3.7 ± 0.3 Hz when mean membrane potential was increased from 4 mV below threshold to above spike threshold (t5 = 4.4, P < 0.01) and oscillation power was increased from 0.81 ± 0.20 mV2/Hz to 2.84 ± 0.14 mV2/Hz at depolarized membrane potentials (Fig. 1C; t5 = 8.4, P < 0.01). Peak-to-peak amplitude of oscillations was increased from 1.3 ± 0.1 to 3.6 ± 0.4 mV.
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To verify that oscillations in LM cells were not dependent on glutamate or GABAA synaptic inputs from other cells, recordings were repeated during blockade of non-N-methyl-D-aspartate (non-NMDA), NMDA, and GABAA synaptic transmission with CNQX (20 µM), AP5 (50 µM), and bicuculline (25 µM), respectively. Oscillations were not reduced in power or frequency in the presence of these antagonists (n = 12; Fig. 2) (2.03 ± 0.30 mV2/Hz and 3.2 ± 0.3 Hz in ACSF, 1.86 ± 0.25 mV2/Hz and 3.5 ± 0.3 Hz in antagonists), indicating that the generation of oscillations does not require glutamate or GABAA synaptic input from other cells.
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To determine if the frequency of oscillations approaches that of the
endogenous theta (4-12 Hz) EEG rhythm at more physiological temperatures, recordings were repeated with the temperature of the bath
increased from 22 to 32°C (n = 5; Fig.
3). Raising temperature reduced spike
duration (2.4 ± 0.3 to 1.2 ± 0.1 ms) and amplitude (91 ± 2 to 76 ± 3 mV), consistent with an increase in the kinetics of K+ conductances (Shen and Schwartzkroin
1988; Thompson et al. 1985
). Raising temperature
significantly increased oscillation frequency from 3.2 ± 0.3 Hz
to 7.1 ± 0.7 Hz (t4 =
4.9,
P < 0.01) at membrane potentials near threshold while
having no significant effect on the power of oscillations (1.92 ± 0.22 mV2/Hz at 22°C, 2.08 ± 0.50 mV2/Hz
at 32°C; Fig. 3C). Action potentials maintained a
clustered firing pattern at 32°C, and additional modest current
injection induced sustained patterns of firing at frequencies between 9 and 14 Hz. At more physiological temperatures, the frequency of oscillations is therefore similar to that of the endogenous 4-12 Hz
theta rhythm (Petsche et al. 1962
).
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Oscillations do not require inward Ca2+ currents
Low-threshold Ca2+ spikes contribute to membrane
potential oscillations in thalamic and mammillary complex neurons
(Alonso and Llinás 1992; Jahnsen and
Llinás 1984
), and low-threshold transient Ca2+ currents have been reported in LM interneurons
(Fraser and MacVicar 1991
). The potential role of
Ca2+ and Ca2+-dependent K+
conductances (Sah 1996
) in the generation of
oscillations in LM cells was tested by monitoring the effects of the
Ca2+ channel blocker Cd2+ (50 µM;
n = 4) and Ca2+-free ACSF
(n = 6; Fig. 4).
Amplitudes of afterhyperpolarizations, which are dependent on
Ca2+-activated K+ conductances, were reduced by
Cd2+ (fAHP, 8.6 ± 1.4 mV to 6.4 ± 0.9 mV; mAHP,
5.0 ± 1.7 mV to 2.3 ± 1.5 mV; Fig. 4B) and by
Ca2+-free ACSF (fAHP, 7.0 ± 1.7 mV to 1.4 ± 0.5 mV; mAHP, 9.7 ± 1.8 mV to 4.2 ± 1.4 mV). There was a small
increase in action potential duration in Ca2+-free ACSF
(2.9 ± 0.2 ms to 4.1 ± 0.3 ms) but not in Cd2+
(2.6 ± 0.3 ms to 2.5 ± 0.3 ms). The frequency and amplitude
of membrane potential oscillations, however, were unaffected by either Cd2+ or Ca2+-free ACSF (Fig. 4C).
Neither treatment caused significant changes in either the frequency
(112 ± 16% of control in Cd2+; 101 ± 7% of
control in Ca2+-free ACSF) or power (105 ± 4% of
control in Cd2+; 89 ± 28% of control in
Ca2+-free ACSF) of oscillations. Therefore neither
Ca2+ currents nor Ca2+-dependent K+
currents appear to be required for membrane potential oscillations in
LM cells.
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Oscillations require sodium currents
Inward Na+ currents play an essential role in
generating theta-frequency oscillations in the entorhinal cortex
(Alonso and Llinás 1989; Klink and Alonso
1993
). The role of Na+ conductances in oscillations
in LM cells therefore was tested using the Na+ channel
blocker TTX. Bath application of TTX (0.5 µM) eliminated Na+-dependent action potentials evoked by depolarizing
current pulses and also totally eliminated membrane potential
oscillations in LM cells (n = 7; Fig.
5). Membrane potential oscillations in LM cells therefore are dependent on inward Na+ currents for
the generation of the depolarizing phase of the oscillations. Further,
because oscillations in LM cells are observed at subthreshold membrane
potentials in the absence of sustained repetitive spiking, they appear
dependent on a persistent (noninactivating) Na+ current
(Klink and Alonso 1993
).
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Oscillations do not require the hyperpolarization-activated current Ih
Regular, repetitive firing properties of interneurons in stratum
oriens-alveus of the CA1 region (Maccaferri and McBain
1996a) and of neurons in the inferior olive (Bal and
McCormick 1997
) are determined in part by the
hyperpolarization-activated mixed cationic current
Ih (Hotson et al. 1979
). To
determine if Ih is involved in membrane
potential oscillations in LM interneurons, recordings were conducted in
the presence of 1 mM Cs+ to block Ih
(n = 4; Fig. 6). The
voltage- and time-dependent inward rectification of responses to
hyperpolarizing current pulses was eliminated by bath application of
Cs+, and the steady-state input resistance measured at the
end of current pulses was increased from 231 ± 7 to 431 ± 131 M
(Fig. 6B). Action potentials and
afterhyperpolarizations were unaffected by Cs+. Although
Cs+ effectively blocked inward rectification in LM cells,
it had no significant effect on either the frequency (3.7 ± 0.6 Hz in ACSF vs. 3.4 ± 0.2 Hz in Cs+) or power
(1.48 ± 0.08 mV2/Hz in ACSF vs. 1.86 ± 0.34 mV2/Hz in Cs+) of membrane potential
oscillations (Fig. 6A). These results, coupled with the
observation of oscillations in LM cells that did not display
significant inward rectification (data not shown), indicate that
Ih is not necessary for the oscillations and
does not contribute significantly to their pacing.
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Potassium currents
Theta-frequency membrane potential oscillations in entorhinal
cortex stellate cells are generated by an interaction between a
persistent Na+ current and outward K+ currents
(Klink and Alonso 1993), and oscillations in LM cells occur within a voltage range in which many K+ currents are
activated. The role of K+ conductances in oscillations in
LM cells therefore was tested using known K+ channel
blockers. First, the effects of the widely acting K+
channel blocker Ba2+ were examined (Fig.
7). Block of K+ conductances
by Ba2+ (1 mM; n = 7) led to membrane
potential depolarization, and negative current injection was used to
test cells at the same membrane potentials. Action potential duration
was increased from 3.1 ± 0.3 to 4.7 ± 0.5 ms, and amplitude
was reduced for both fAHPs (7.4 ± 1.6 to 2.9 ± 1.5 mV) and
mAHPs (8.1 ± 0.5 to 4.5 ± 1.1 mV) in Ba2+ (Fig.
7B). Barium also caused significant reductions in
oscillation power (45 ± 10% of control;
t6 = 4.4, P < 0.01) and
frequency (58 ± 7% of control; t6 = 4.8, P < 0.01) at membrane potentials near spike threshold
(e.g., Fig. 7A). The hyperpolarizing phase of oscillations
in LM cells therefore may result from the activation of
voltage-dependent K+ currents sensitive to
Ba2+.
|
To characterize in more detail the K+ currents that mediate
the repolarizing phase of oscillations, the effects of high and low
doses of the K+ channel blocker 4-AP (10 mM,
n = 5; 50 µM, n = 6) were examined. A
slowly inactivating delayed-rectifying K+ current in
cultured neonatal LM cells (Chikwendu and McBain 1996) and the slowly inactivating K+ current
ID (Storm 1990
) are sensitive to
low doses of 4-AP, whereas high doses of 4-AP block the transient
current IA (Storm 1990
). Both
high and low doses of 4-AP had strong effects on oscillations and
electrophysiological properties of LM cells (Fig.
8). The low concentration of 4-AP (50 µM) increased spike duration (2.9 ± 0.3 to 5.0 ± 0.6 ms)
and reduced the amplitude of fAHPs (9.9 ± 0.4 to 5.0 ± 1.8 mV) and mAHPs (8.7 ± 2.2 to 3.1 ± 1.3 mV) (e.g., Fig.
8B). The high dose of 4-AP (10 mM) also reduced action
potential repolarization and eliminated afterhyperpolarizations (data
not shown). Both high and low doses of 4-AP significantly reduced the
frequency (57 ± 11% of control for 10 mM; 63 ± 6% of
control for 50 µM; F1 = 29.8, P < 0.001) and power (51 ± 9% of control for 10 mM; 26 ± 4% of control for 50 µM; F1 = 17.0, P < 0.01) of oscillations in LM cells at
membrane potentials near threshold. Effects of 10 mM 4-AP were
not significantly different from those of 50 µM 4-AP (frequency,
F1,1 = 1.6, P = 0.24; power,
F1,1 = 4.1, P = 0.07; Fig.
8C).
|
The contributions of K+ conductances sensitive to TEA
to the oscillations were examined using high (30 mM) and low (100 µM) doses of TEA. Low doses of TEA preferentially block a sustained delayed-rectifier K+ current in neonatal LM cells, whereas
high doses of TEA block in addition a slowly inactivating
delayed-rectifier current (Chikwendu and McBain 1996).
Although both high and low doses of TEA had strong effects on
electrophysiological properties of LM cells, membrane potential
oscillations persisted in the presence of both concentrations of TEA
(Fig. 8). Thirty millimolar TEA increased spike duration (3.1 ± 0.2 to 18.5 ± 5.4 ms), eliminated fAHPs, and reduced the
amplitude of mAHPs (9.7 ± 1.3 to 1.7 ± 1.1 mV) (Fig.
9B). The 100 µM dose of TEA
reduced the amplitude of fAHPs (12.9 ± 2.3 to 10.1 ± 2.6 mV) and mAHPs (11.1 ± 1.6 to 8.4 ± 1.8 mV) but did not
significantly increase spike duration (2.1 ± 0.1 to 2.7 ± 0.5 ms). Oscillations in membrane potential were observed reliably in
the presence of both 30 mM and 100 µM TEA at membrane potentials near
threshold, and neither dose caused a significant reduction in either
the frequency (108 ± 10% of control in 100 µM TEA; 85 ± 7% of control in 30 mM TEA) or power (109 ± 24% of control in
100 µM TEA; 132 ± 12% of control in 30 mM TEA) of oscillations (Fig. 9, A and C). The K+
conductances involved in oscillations in LM cells therefore appear insensitive to high and low doses of TEA.
|
Membrane potential oscillations in stratum oriens interneurons
To examine if membrane potential oscillations similar to
those found in LM interneurons also were present in other types of interneurons, cells in stratum oriens were examined under similar conditions. The electrophysiological properties of interneurons located
in stratum oriens were more heterogeneous than those of LM interneurons
(Freund and Buzsáki 1996; Lacaille and
Williams 1990
; Maccaferri and McBain 1995
,
1996b
) (e.g., Fig.
10A), and only one of eight
cells showed membrane potential oscillations clearly in the same
frequency range as LM cells. Two cells had action potentials of
short-duration (1.1 and 1.3 ms) and prominent fAHPs (8.4 and 14.8 mV;
e.g., Fig. 10A2). Both of these cells showed regular,
voltage-dependent oscillations, but the frequency of the oscillations
was higher than that of LM cells (5.9 and 42 Hz at 22°C, e.g., Fig.
10B2). A third interneuron with long-duration action
potentials (2.7 ms) showed oscillations that also were higher in
frequency (7.3 Hz) than in LM cells and that persisted in the presence
of CNQX, AP5, and bicuculline. Of five remaining interneurons tested in
the presence of these antagonists (action potential duration, 2.0-3.3
ms), only one showed regular membrane potential oscillations similar to
those observed in LM cells (not shown). The membrane potential of the
other four interneurons fluctuated more near threshold than at
hyperpolarized levels, but regular, rhythmic oscillations were not
observed (compare Fig. 10B, 1 and 3).
|
Morphological information was obtained from 65 biocytin-filled
interneurons in LM and 7 in stratum oriens. The morphology of cells in
LM was similar to that reported previously (Kunkel et al.
1988; Morin et al. 1996
; Williams et al.
1994
) (Fig. 10C). Somata of LM cells (10-25 µm
diameter) were usually fusiform (n = 42 cells) and
oriented parallel to the pyramidal cell layer (35 of 42 cells), but
some cells with multipolar somata also were observed (n = 12 cells). They usually had two to three primary dendrites, which
bifurcated close to the soma and arbourized in LM and stratum radiatum
(n = 64 cells). Axons of LM cells tended to originate
from a primary dendrite and arbourized mostly in LM and stratum
radiatum (n = 38 cells) but also were observed in
stratum pyramidale and stratum oriens (n = 11 cells).
Somata of interneurons located in stratum oriens were fusiform
(n = 4 cells) or multipolar (n = 2 cells) with two to six primary dendrites which arbourized in stratum
oriens and the alveus (n = 7 cells), and also sometimes
in stratum radiatum (n = 3 cells; Fig. 10D). Axon collaterals were observed in stratum oriens (n = 3 cells) and also projected to stratum radiatum in one cell.
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DISCUSSION |
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Membrane potential oscillations in LM interneurons of the CA1
region were characterized and shown to arise from intrinsic voltage-dependent conductances. Oscillations regulate the timing of
cell firing when LM cells are depolarized near threshold. Raising temperature from 22 to 32°C increased oscillation frequency from 3.2 to 7.1 Hz, which falls within the range of the hippocampal theta rhythm
(Petsche et al. 1962). Oscillations were not dependent on synaptic inputs and were eliminated by holding cells 6-10 mV below
threshold, indicating that oscillations are generated by intrinsic
voltage-dependent conductances. Oscillations were not affected by
blocking either Ca2+ currents or Ih
(Alonso and Llinás 1992
; Maccaferri and
McBain 1996a
) but were blocked by TTX and reduced by the
K+ channel blockers Ba2+ and 4-AP. Oscillations
therefore likely involve an interplay between a persistent
voltage-gated Na+ current and outward K+
currents, similar to that which generates theta-frequency oscillations in stellate cells of the entorhinal cortex (Alonso and
Llinás 1989
; Klink and Alonso 1993
).
Membrane potential oscillations generated by these conductances provide
a mechanism through which depolarization of LM interneurons can lead to
rhythmic inhibition of pyramidal neurons during theta activity.
A high-proportion of LM interneurons displayed theta-frequency membrane
potential oscillations, but these were much less common in interneurons
in stratum oriens. Most interneurons in stratum oriens either did not
show oscillations in membrane potential or displayed oscillations that
occurred at higher frequencies than in LM cells. The tendency of
stratum oriens interneurons to oscillate at higher peak frequencies
than LM interneurons suggests that the two classes of interneurons may
contribute preferentially to different frequencies of rhythmic
hippocampal population activity. For example, the higher-frequency
oscillations in interneurons in stratum oriens may contribute to
network interactions thought to underlie gamma frequency (40 Hz)
activity in the CA1 region (Wang and Buzsáki 1996
;
Whittington et al. 1995
). Further,
GABAA-mediated synaptic inhibition of pyramidal cells by LM
interneurons decays more slowly than inhibition mediated by other
interneuron subtypes (
d
40 ms) (Banks et al.
1998
; Ouardouz and Lacaille 1997
) and may allow
LM cells to contribute most effectively to slower frequencies of
postsynaptic rhythmic activity. Therefore both intrinsic conductances underlying interneuron membrane potential oscillations and different time courses of synaptic inhibition may be important for generating different frequencies of rhythmic population activity.
Conductances generating oscillations
The persistence of membrane potential oscillations in blockers of
ionotropic glutamatergic and GABAergic synaptic transmission indicates
that intrinsic membrane conductances generate oscillations in LM cells.
Oscillations were preserved in Ca2+-free ACSF and in
Cd2+ that blocked voltage-dependent Ca2+
currents (Fig. 4). Calcium currents and Ca2+-dependent
K+ currents (Fraser and MacVicar 1991;
Sah 1996
) that contribute to oscillations in the
thalamus and mammillary complex (Alonso and Llinás
1992
; Jahnsen and Llinás 1984
) are
therefore not required for the generation of theta-frequency
oscillations in LM cells. Many LM cells have inwardly rectifying
voltage responses to hyperpolarizing current pulses; this suggested
that the hyperpolarization-activated cationic current
(Ih), which paces cell firing in CA1
oriens/alveus interneurons (Macafferri and McBain 1996a
)
and inferior olive neurons (Bal and McCormick 1997
),
might contribute. However, Ih normally was
activated at membrane potentials more negative than the voltage range
in which oscillations were observed (see also Macafferri and
McBain 1996a
) and oscillations were not affected by blocking
Ih with Cs+. Further, oscillations
also were observed consistently in cells without inwardly rectifying
I-V responses. Therefore Ih does not contribute significantly to the pacing of membrane potential
oscillations in LM interneurons.
The effects of Na+ and K+ channel blockers on
oscillations in LM cells indicate that oscillations result from an
interplay between voltage-dependent Na+ and K+
conductances. Oscillations were eliminated by the Na+
channel blocker TTX and strongly reduced in amplitude and frequency by
the K+ channel blockers Ba2+ and 4-AP. Each
oscillation cycle, which begins when cells are depolarized near spike
threshold, therefore likely is generated by activation of a persistent
Na+ current (INa(p)) followed by the
activation of repolarizing K+ currents. Similar mechanisms
mediate theta-frequency oscillations in stellate cells of the
entorhinal cortex (Klink and Alonso 1993). Sodium
(Garcia-Munoz et al. 1993
; Leung and Yim
1991
) and potassium (Leung and Yim 1991
)
conductances also are implicated in intrinsic membrane potential
oscillations in CA1 pyramidal neurons, which, like oscillations in LM
cells, are not sensitive to Ca2+-free ACSF,
Cs+, or Cd2+ (Garcia-Munoz et al.
1993
; Leung and Yim 1991
). Because
INa(p) is noninactivating, oscillation frequency
in LM cells may be governed by the inactivation time constant of the
K+ current. Therefore the voltage-dependent K+
current must inactivate over roughly one-half of the period of the
theta rhythm. Increased K+ channel inactivation kinetics
likely account for the increase in oscillation frequency from 3 to 7 Hz
when temperature was raised from 22 to 32°C.
The present results indicate that the K+ conductances
underlying oscillations in LM cells are sensitive to Ba2+
and 4-AP but not TEA. The classic transient K+ current
IA (Storm 1990) has an
appropriate inactivation time constant (
d = 50 ms) but is not blocked by 50 µM 4-AP and therefore cannot
alone support oscillations. Transient K+ currents have been
observed in LM cells, but they require strong hyperpolarization to
deinactivate (Fan and Wong 1996
). Potassium conductances
generating oscillations in LM cells must inactivate and recover from
inactivation rapidly in the absence of strong hyperpolarization because
oscillations were observed regularly in the absence of
afterhyperpolarizations evoked by cell firing (e.g., Figs. 1 and 3).
The delay current ID is sensitive to low concentrations of 4-AP (Storm 1990
) similar to those
that blocked oscillations, but ID inactivation
requires seconds and is too slow to mediate theta-frequency
oscillations. Two voltage-dependent delayed-rectifying K+
currents have been identified in cultured neonatal LM cells: a slowly
inactivating current sensitive to low concentrations of 4-AP and a
sustained current sensitive to low concentrations of TEA
(Chikwendu and McBain 1996
). Oscillations cannot be
mediated by the sustained current because they persisted in TEA and
were blocked by 4-AP which does not reduce the sustained current. The sensitivity of the slowly inactivating current to low concentrations of
4-AP (Chikwendu and McBain 1996
) suggested that it may
contribute significantly to oscillations, but most of this current also
is blocked by high doses of TEA (Chikwendu and McBain
1996
), which did not block oscillations. These findings suggest
that K+ currents in LM interneurons of the mature
hippocampus may have different pharmacological sensitivities than those
described for neonatal LM cells (Chikwendu and McBain
1996
) and that K+ conductances mediating
theta-frequency oscillations in the adult may not be present in
neonatal LM interneurons. The less frequent observation of membrane
potential oscillations in LM cells of younger rats (Williams et
al. 1994
) is consistent with a developmental regulation of
K+ conductances mediating oscillatory activity in LM interneurons.
Genesis and phase-locking of oscillations
LM interneurons are typically silent at resting membrane potential
and must be depolarized near spike threshold for membrane potential
oscillations to be expressed. The medial septum is likely to play a
central role in the depolarization and induction of membrane potential
oscillations in LM cells. Cholinergic afferents from the medial septum
synapse onto LM interneurons (Frotscher and Léránth
1985) and CA1 interneurons are depolarized by cholinergic agonists (Behrends and Ten Bruggencate 1993
;
Reece and Schwartzkroin 1991
). In the entorhinal cortex,
membrane potential oscillations in stellate cells are induced by
cholinergic depolarization (Klink and Alonso 1997
).
To contribute significantly to rhythmic inhibition of pyramidal cells
during theta activity, the repetitive firing of a large number of LM
cells must be closely phase-locked; randomly phased LM cell firing
would result in tonic hyperpolarization of pyramidal neurons. LM cells
mediate primarily feedforward inhibition in CA1 (Lacaille and
Schwartzkroin 1988) so that feedback excitation by pyramidal
neurons during theta activity is likely insufficient for their
synchronization. However, excitatory input to LM interneurons from CA3
pyramids or contralateral CA1 region (Kunkel et al.
1988
), rhythmic inhibitory GABAergic inputs from the medial
septum (Petsche et al. 1962
; Tóth et al.
1997
), and mutual inhibition among interneurons (Atzori
1996
) also may synchronize the phase of LM cell oscillations with respect to theta activity in pyramidal cells. Finally, because direct inputs to stratum lacunosum-moleculare from the entorhinal cortex terminate on both interneurons and pyramidal cells
(Desmond et al. 1994
; Kunkel et al.
1988
), theta-frequency input from entorhinal cortex neurons
(Mitchell and Ranck 1980
) might rhythmically excite and
synchronize many LM cells. Thus entorhinal cortex afferents to LM cells
indirectly may control the phase of output of CA1 pyramidal cells to
parahippocampal areas, including the entorhinal cortex.
In conclusion, LM cells of the CA1 region possess intrinsic Na+ and K+ conductances that generate theta-frequency oscillations in membrane potential near threshold. LM interneurons therefore likely contribute to hippocampal theta activity by rhythmically inhibiting and synchronizing pyramidal neurons. Local hippocampal circuits and extrahippocampal afferents may thus contribute to hippocampal theta activity by an action on LM interneurons.
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ACKNOWLEDGMENTS |
---|
The authors thank Dr. Chris J. McBain for helpful discussions.
This research was funded by Grant MT-10848 to J.-C. Lacaille from the Medical Research Council of Canada. J.-C. Lacaille is a senior scholar with the Fonds de la Recherche en Santé du Québec, a member of the Groupe de Recherche sur la Système Nerveux Central [Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR)], and a member of an Equipe de recherche from FCAR. C. A. Chapman was supported by postdoctoral fellowships from the Centre de Recherche en Sciences Neurologiques (J-P Cordeau Fellowship) and the Natural Sciences and Engineering Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests: J.-C. Lacaille, Département de Physiologie, Faculté de Médecine, Université de Montréal, C.P. 6128 Succ. Centre-ville, Montreal, Quebec H3C 3J7, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 July 1998; accepted in final form 9 November 1998.
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REFERENCES |
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