Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Sun, Miao-Kun,
Wei-Qin Zhao,
Thomas J. Nelson, and
Daniel L. Alkon.
Theta Rhythm of Hippocampal CA1 Neuron Activity: Gating by
GABAergic Synaptic Depolarization.
J. Neurophysiol. 85: 269-279, 2001.
Information processing and memory
consolidation during exploratory behavior require synchronized activity
known as hippocampal theta () rhythm. While it is well established
that the
activity depends on cholinergic inputs from the medial
septum/vertical limb of the diagonal band nucleus (MS/DBv) and
discharges of GABAergic interneurons, and can be induced with
cholinergic receptor agonists, it is not clear how the increased
excitation of pyramidal cells could occur with increased discharges of
GABAergic interneurons during
waves. Here, we show that the
characteristic
activity in adult rat hippocampal CA1 pyramidal
cells is associated with GABAergic postsynaptic depolarization and a
shift of the reversal potential from Cl
toward
HCO3
(whose ionic gradient is regulated by carbonic
anhydrase). The
activity was abolished by
GABAA receptor antagonists and carbonic anhydrase
inhibitors, but largely unaffected by blocking glutamate receptors.
Carbonic anhydrase inhibition also impaired spatial learning in a
watermaze without affecting other sensory/locomotor behaviors. Thus
HCO3
-mediated signaling, as regulated by carbonic
anhydrase, through reversed polarity of GABAergic postsynaptic
responses is implicated in both
and memory consolidation in rat
spatial maze learning. We suggest that this mechanism may be important
for the phase forward shift of the place cell discharges for each
cycle during the animal's traversal of the place field for that cell.
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INTRODUCTION |
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Synchronization of neural
activity within mammalian brain structures, as occurs during
hippocampal rhythm (O'Keefe and Recce 1993
;
Shen et al. 1997
; Skaggs and McNaughton
1996
), contributes to diverse forms of information coding
(Draguhn et al. 1998
; Rodriguez et al.
1999
; Usher and Donnelly 1998
). The
frequency field oscillation, a major feature of the hippocampal
electroencephalogram (EEG), for example, occurs during two specific
behaviors, exploration and rapid-eye-movement (REM) sleep, and reflects
synchronized synaptic potentials that entrain the discharge of neurons
at frequencies between 4 and 12 Hz. The rhythm is believed by many to
gate or facilitate memory information processing in the hippocampus,
particularly during persistent information storage. Thus as an animal
explores its environment, MS/DBv cholinergic inputs, which innervate
the whole hippocampal formation (Dutar et al. 1995
;
Vertes and Kocsis 1997
), activate hippocampal
rhythm
(Vertes and Kocsis 1997
). Briefly increased
power
has been reported during a word recognition memory task in humans, with
a delay of about 125 ms after the visual presentation of a word
(Burgess and Gruzelier 1997
). Recording neuromagnetic
signals during a working memory task in humans reveals stimulus-locked
hippocampal
(Tesche and Karhu 2000
). Evidence has
also been provided that disruption of the
activity by lesions of
cholinergic inputs to the hippocampus blocks spatial memory (Winson 1978
). The synaptic bases of the
rhythm have
been extensively studied, but many important questions related to the
underlying mechanism(s) for the
activity remain to be answered. For
instance, while the cholinergic
activity recorded in place
pyramidal cells is known to depend on
rhythmic activity from
GABAergic interneurons, pyramidal cells are excited when the animals
travel into the field of the place cell, i.e., when GABAergic
interneurons are most active (Cscsvari et al. 1999
;
Soltesz and Deschenes 1993
; Ylinen et al.
1995
). Furthermore, the firing period of the place cell during
the exploration traversal shifts forward during each
wave and
becomes more in phase with interneuron discharge.
The cholinergic activity in the hippocampus can be induced in vitro
(e.g., Huerta and Lisman 1995
; Pitler and Alger
1992
; Vertes and Kocsis 1997
), while it remains
to be established to what extent such induced
activity in vitro
represents the
rhythm EEG in behaving animals. We report here that
cholinergic
activity in hippocampal CA1 pyramidal cells involves a
switch of GABAergic postsynaptic responses from a predominantly
hyperpolarizing Cl
to a depolarizing,
predominantly HCO3
conductance. GABAergic activity
through the reversed polarity can effectively and immediately entrain
the pyramidal cells into a
rhythm. Reducing HCO3
formation by inhibition of carbonic anhydrase blocks
rhythm induction in vitro and impairs rat watermaze performance in vivo. Switching between these operational states of the synapses may thereby
provide a powerful way to selectively direct signal processing through
the network.
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METHODS |
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Chemicals
Agents were either injected into the recorded cells through the
recording electrodes: benzolamide (gift from T. H. Maren, University of Florida, Gainesville; 0.1 mM; 0.5 nA, 500 ms at 50% on
cycles for 10 min) and calexcitin (260 ng/µl of cloned calexcitin in
1 M K acetate, pH 7.4; 2.0 nA, 700 ms at 33% on cycles for 15 min),
or through the perfusion medium: kynurenic acid (Sigma), bicuculline
methiodide (BIC; Sigma); carbachol (CCH; Sigma), acetazolamide (ACET;
Sigma), and atropine sulfate (Sigma).
Hippocampal slice electrophysiology
CA1 field potentials were recorded with glass microelectrodes
filled with an artificial cerebrospinal fluid solution (ACSF; see
below). Male Sprague-Dawley rats (150-200 g) were decapitated, and the
brains were removed and cooled rapidly in an ACSF solution (~4°C),
bubbled continuously with 95% O2-5%
CO2. Hippocampi were sliced (400 µM), placed in
oxygenated ACSF (in mM: 124 NaCl, 3 KCl, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose), and perfused (2 ml/min) with the oxygenated ACSF in an interface chamber at 30-31°C. Whole slices were used unless otherwise
indicated. CA1 pyramidal cells were recorded intracellularly
(Sun et al. 1999 for cell labeling) with sharp
electrodes (3 M KAc; tip resistance: 60-120 M
; to prevent
"run-down" of GABAergic responses in whole cell recordings due to
wash out of intracellular factors). Stable GABAergic inhibitory
postsynaptic response (IPSP) could thus be evoked for several hours
without noticeable change in amplitudes. Signals were amplified with
AxoClamp-2B amplifier, digitized, stored, and analyzed using DigiData
1200 with P-Clamp6 software (Axon Instruments). Frequency and amplitude
values of oscillation were taken from an average of 5 consecutive
traces, all triggered at the same level of the same phase. Capacitance
was optimally adjusted during discontinuous current-clamp mode before
and after cell penetration to neutralize capacitance and reduce
overshoot/undershoot errors. Discontinuous single-electrode
voltage-clamp mode was used for voltage-clamping, employing a sampling
rate of 3.0-5.0 kHz (30% duty cycle). Gain was usually set at 6-8 nA
· mV
1, slightly below
the maximum value without causing overshoot or instability in the step
response to a repetitive 10-mV step command. Bipolar stimulating
electrodes (Teflon-insulated PtIr wire with 25 µm diam) were placed
in s. pyramidale, within 200 µm from the recording electrode, for
stimulation of interneurons (50 µA, 50 µA) in the pyramidale layer.
In some cases, the position of the stimulating electrodes was slightly
varied within the CA1 cell layer to obtain monophasic postsynaptic
responses. Test stimuli were applied at 1 per minute (0.017 Hz). In
some experiments, an additional stimulating electrode was placed in the
stratum radiatum to stimulate the Schaffer collateral pathway (Sch).
Experiments in which >20% variations in the evoked IPSP magnitudes
occurred during the 10-min control period were discarded.
Spatial maze tasks
Effects of reducing HCO3 formation in
vivo on spatial memory were evaluated in rats with Morris watermaze
task (Meiri et al. 1998
). Male adult Wistar rats were
housed in a temperature-controlled (20-24°C) room for 1 wk, allowed
free access to food and water, and kept on a 12-h light/dark cycle. On
the first day of experiments, all rats were randomly assigned to
different groups (10 each) and swam for 2 min in a 1.5-m
(diameter) × 0.6-m (depth) pool (22 ± 1°C). On the
following day, rats were trained in a 4 trial/day task for 4 consecutive days. Each training trial lasted for up to 2 min, during
which rats learned to escape from water by finding a hidden platform
placed in a fixed location and submerged about 1 cm below the water
surface. A quadrant test was performed after removing the platform
24 h after the last training trial. The route of rats' swimming
across the pool was recorded. The number of grid crossings on record
paper in each quadrant was counted and used as arbitrary swimming
distance units. A single dose of ACET (5 mg/0.5 ml saline/day, freshly
prepared) was injected (intraperitoneal), about 65-70 min prior to the
first trial or quadrant test. The control rats received the same volume
(intraperitoneal) of saline.
Statistical analysis was performed using the Student's t-test for paired or unpaired data or ANOVA whenever appropriate. The values are expressed as means ± SE of the mean, with n indicating number of the cells or rats. All animals used in these experiments were treated under National Institutes of Health guidelines for the welfare of laboratory animals.
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RESULTS |
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CCH-induced field oscillation and intracellular
To simulate cholinergic septal activation and diffuse
acetylcholine transmission (Descarries et al. 1997), we
bath applied CCH (50 µM, 20 min), a cholinergic receptor agonist, to
hippocampal slices from adult rats. CCH triggered a local
field
potential (Fig. 1A; peak
amplitude: 0.75 ± 0.03 mV, mean ± SE, n = 12, P < 0.05 from background noise; at 7.8 ± 0.8 Hz; n = 12), lasting for the post-CCH recording period
of ~3 h (Huerta and Lisman 1995
; Pitler and
Alger 1992
). The
activity varied in magnitude, indicating summation of different numbers of neurons discharging in each phase.
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The activity was blocked by bath atropine sulfate (1 µM,
n = 6; not shown), a muscarinic antagonist, as reported
by others (e.g., Huerta and Lisman 1995
), and was
generated in the CA1. The CCH-induced activity (0.73 ± 0.04 mV,
n = 7, P < 0.05; at 7.7 ± 0.9 Hz; n = 7, P < 0.05) in CA1
minislices, after dissecting away both CA3 and dentate gyrus, did not
differ (P > 0.05; unpaired t-test) from
that of the whole slices. The
oscillation frequency did not change
(n = 8, P > 0.05), although the
oscillation magnitude was slightly reduced, in the presence of
kynurenate, an N-methyl-D-aspartate (NMDA)- and
non-NMDA receptor antagonist (Collingridge and Lester 1989
). Kynurenate was applied extracellularly at 500 µM
(20-30 min), a concentration at which it effectively abolished
excitatory postsynaptic responses of CA1 pyramidal cells to stimulation
of the Schaffer collateral pathway (Sun et al. 1999
) or
responses of other brain neurons to L-glutamate (Sun
1996
). CCH induced
oscillation of membrane potential
(7.8 ± 1.1 mV; n = 20; P < 0.05)
in CA1 pyramidal cells (intracellular
; Fig. 1B), a
response blocked by bath atropine sulfate (1 µM, n = 8, P < 0.05; not shown). At one-third to one-half of
the maximum depolarizing phase, action potentials were triggered (Fig.
1, B and C). During a 5-min observing period, CCH
induced an averaged discharge rate of 2.7 ± 0.3 spikes/s, significantly higher (n = 20, P < 0.05) than their pre-CCH rate (0.0 ± 0.0 spikes/s). These
variations were consistent with those of the field
magnitude
recorded. The intracellular
remained unchanged when the membrane
potential of the cells was maintained at their pre-CCH levels.
Involvement of GABAergic postsynaptic depolarization in the CA1
activities
Bath-applied BIC (1 µM) eliminated the field oscillation (by
97.5 ± 4.2%, n = 8, P < 0.05;
Fig. 1A) and CA1 intracellular
activity (by 98.9 ± 3.4%, n = 10, P < 0.05; Fig.
1B). When applied before the CCH application, BIC did not
produce obvious changes in the field potential (n = 6)
or membrane potentials of CA1 pyramidal cells (n = 8),
but prevented CCH effects on the
activity induction. At 1 µM, BIC
did not produce any obvious excitation of the CA1 cells. Activation of
the GABAA receptors is thus necessary for CCH to
elicit synchronous CA1 field events. Suppressing
GABAA receptor channels alone is insufficient to
induce
.
The GABAergic inputs were activated by microstimulation of s.
pyramidale. The evoked IPSPs in CA1 pyramidal cells depended on the
membrane potentials (e.g., Fig. 3). Thus the IPSPs were always
monitored with values compared at their pretest control membrane
potentials. The evoked IPSPs (Fig.
2A; peak response: 8.89 ± 0.29 mV, n = 89) were not altered by kynurenate (500 µM, n = 6), but abolished by BIC (1 µM; by
96.8 ± 3.7%; n = 8, P < 0.05),
indicating GABAA receptor mediation and an
absence of contamination of any obvious excitatory component in the
evoked IPSPs. Associated with the
activity was a gradual reduction
in the IPSPs (n = 25) and the ultimate production of an
"excitatory" response (Fig. 2, A and B; from
pre-CCH
9.0 ± 1.2 mV as compared with +5.1 ± 0.4 mV 30 min after the CCH application; n = 10, P < 0.05). This excitatory response was observed at
the pre-CCH membrane potential maintained by intracellular injection of
hyperpolarizing current. These voltage changes in the GABAergic
responses corresponded to a gradual change of an outward current
(0.18 ± 0.03 nA) toward an inward current (0.19 ± 0.05 nA;
n = 5, P < 0.05) under voltage clamp
(Fig. 2C). The intracellular
activity became evident
when the GABAergic responses became depolarizing (Fig. 2A).
Measured when the
activity became evident, the input resistance
(79.2 ± 1.6 M
) of the cells did not significantly differ
(n = 10; P > 0.05) from their pre-CCH
value (80.5 ± 1.4 M
). Depressing GABAA responses alone was insufficient to induce the
activity since BIC
did not induce the rhythmic activity (see last paragraph). The
reversed excitatory response was also sensitive to BIC (Fig. 2B), indicating the involvement of the same type of receptor
channel before and after the CCH administration.
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The relationship between the maximum responses of hippocampal CA1
pyramidal cells to stimulation of the GABAergic inputs and membrane
potential at which the inputs were activated can be described with a
straight line. BIC virtually abolished the GABAergic postsynaptic responses no matter whether the postsynaptic responses were evoked at
membrane potentials positive or negative to the reversal potential (Fig. 3A). The reversal
potential, however, was not changed by BIC (Fig. 3A;
81.3 ± 2.6 mV; n = 6). This BIC effect
contrasts with CCH-induced changes that were associated with a positive shift of the reversal potential (Fig. 3B; from
79.8 ± 3.2 to
68.4 ± 2.8 mV; n = 10, P < 0.05). Thus the CCH-induced changes in GABAergic
responses are fundamentally distinct from a reduced response and could
not result from a diminished GABAergic synaptic transmission
(suppressed presynaptic release or postsynaptic response). Figure
3C illustrates an example in which the CCH-induced reversal potential appears to be above the threshold (approximately
57 mV) for
generation of action potential. Thus single brief pulse of stimulation
of the GABAergic inputs elicited action potential during post-CCH
period in the cell, in contrast to inhibitory postsynaptic response
before the CCH application (Fig. 3C).
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Elimination of CA1 activities and GABA depolarization by
carbonic anhydrase inhibitors
Bath ACET (1 µM), a carbonic anhydrase inhibitor, eliminated the
CCH-induced changes in GABAergic postsynaptic responses (Fig. 4B). The evoked IPSP
(7.7 ± 1.0 mV, n = 12, P < 0.05) in the presence of ACET and CCH did not differ (n = 12, P > 0.05) from their control values (
7.8 ± 1.1 mV). Under such conditions, neither
field oscillation
(n = 8; Fig. 4A) nor intracellular
activity (n = 10; Fig. 4B) was induced by
CCH. Similarly, intracellular application of benzolamide, a
membrane-impermeable carbonic anhydrase inhibitor, prevented the
occurrence of CCH-induced reversed GABAergic responses and
intracellular
activity (n = 6), indicating an involvement of intracellular carbonic anhydrase. Interestingly, application of calexcitin, a memory-related signal protein
(Alkon et al. 1998
; Sun et al. 1999
),
into CA1 pyramidal cells mimicked CCH in inducing the intracellular
activity (Fig. 5, A and
B; n = 10), when associated with a
depolarizing current to load Ca2+. The
calexcitin-induced intracellular
activity was also prevented by
bath ACET (1 µM) in six cells tested (Fig. 5C). These
results indicate a critical role of HCO3
conductance
in an intracellular signaling cascade responsible for the
rhythm.
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Entraining CA1 pyramidal cells by GABAergic inputs
Entraining hippocampal pyramidal cells at frequency has been
proposed to be a fundamental role of the interneurons (Cobb et
al. 1995
; Paulsen and Moser 1998
). How might
GABAergic interneurons entrain the pyramidal cells? The only mechanism
that has been previously proposed is rebound action potential. However,
rebound "depolarization" usually requires resting activity that was
provided by constant current injection (Cobb et al.
1995
), and hippocampal pyramidal cells normally do not show
much spontaneous activity. In some cells (26 of 149 neurons in which
effects of membrane potential changes on the GABAergic postsynaptic
responses were examined), discharges lasted for a period of elicited
depolarization and an evoked IPSP appeared to be able to delay
subsequent spikes (Fig. 6A).
The majority of cells (123 of 149), however, showed a rapid adaptation
to depolarization (Fig. 3, A and B), resulting in
a silent but depolarized state. At resting membrane potential, rebound
depolarization requires very strong hyperpolarization, which naturally
occurring IPSPs are unlikely to provide. No rebound action potential
was observed with IPSPs of
8.9 ± 0.3 mV evoked at resting
membrane potentials (
73.8 ± 0.9 mV, n = 89;
Fig. 6B, trace 1). A train of pulses at 100 Hz
was also ineffective (Fig. 6B, trace 2),
suggesting that temporal summation of the unitary IPSPs is insufficient
to evoke rebound depolarization. Furthermore, no significant rebound
depolarization (0.19 ± 0.12 mV, n = 75, P > 0.05) was evoked with intracellular negative
pulses (up to 700 ms) sufficient to evoke
10.8 ± 1.4 mV
potential changes (Fig. 6C, trace 1) from the
resting membrane potential (
74.8 ± 0.4 mV). In addition, when
evoked at depolarized membrane potentials, the occurrence and timing of
individual "rebound" action potentials varied (Fig. 6D).
Thus rebound action potentials, even when they occur, do not represent
a precise control mechanism. On the other hand, in the presence of CCH,
stimulation of GABAergic inputs elicited instantly phase-locked firing
of pyramidal cells (Fig. 7, A
and C; n = 14). The postsynaptic GABAergic
response to the first stimulation pulse usually did not reach action
potential threshold (Fig. 7, A and C). The
postsynaptic GABAergic responses were sensitive to BIC, indicating the
involvement of the same receptor channels (Fig. 7A). In
eight cells, single pulse stimulation of Sch (10-30 µA, 50 µs)
evoked an excitatory postsynaptic potential of 7.5 ± 1.2 mV,
which was about 50% below the threshold. Before the CCH
administration, co-stimulation of Sch at the set intensity (50% below
the threshold) and GABAergic inputs (50 µA, 50 µs) largely
abolished the Sch stimulation-induced excitatory potential (by
89.5 ± 4.3%, n = 8, P < 0.05;
Fig. 7C). The single-pulse Sch stimulation-evoked excitatory
postsynaptic potential was not altered (P > 0.05) by
CCH (not shown). Action potentials, however, were evoked by
co-stimulation of Sch at below-threshold intensity together with
reversed GABAergic inputs in all cases (n = 8, P < 0.05; Fig. 7C, bottom
right). Thus reversed synaptic responses reshapes the GABAergic
inhibitory function into amplification (Sun et al. 1999
)
and reconfigures the operations of hippocampal networks into patterns
of activity associated with GABAergic inputs (Fig. 7B).
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Spatial memory deficits by ACET administration in vivo
We asked whether reducing HCO3 formation
with a carbonic anhydrase inhibitor that can pass through the blood
brain barrier could affect rat spatial memory. In rats, an
intraperitoneal (ip) dose of ACET produces a peak concentration in the
blood within 1 h and is cleared by 2 h (Cassin et al.
1963
; Sone et al. 1998
). Effects of ACET on
spatial learning (Meiri et al. 1998
) were determined during this short period. A single dose of ACET (14-18 mg/kg, sufficient to reduce the EEG
power by about 50% at maximum during rat REM sleep) (Sone et al. 1998
) was sufficient to
produce memory impairment (Fig.
8A). The ACET group showed a
strikingly smaller reduction (F1,18 = 34.79, P < 0.0001) in escape latency during training
trials than the saline group did. The memory impairment became more
significant as the training days progressed and was particularly
evident in the first trial (65-70 min after the injection) of each
successive day (Fig. 8, A and B). The latter
might reflect a relatively normal short-term (vs. long-term) learning
after ACET or more likely influence of a rapid clearance of the drug (Cassin et al. 1963
; Sone et al. 1998
).
Quadrant tests 24 h after the last training trial revealed that
control rats spent the majority of their time searching in the quadrant
(Quadrant 4; Fig. 8C) where the platform was previously
placed and had been removed (F3,36 = 183.9, P < 0.0001; ANOVA and Newman-Keuls post hoc
test), whereas the ACET group showed no preference to a particular
quadrant (F3,36 = 1.59, P = 0.21; Fig. 8D).
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The total swimming distances, however, did not differ between the two groups (Fig. 8E; P > 0.05), indicating that ACET did not grossly affect their sensory or locomotor activities. Neither was memory retrieval affected by ACET. The control rats were trained for 3 more days (Fig. 9A) and received the single injection of either ACET or saline 24 h after the last training trial. Sixty-five to 70 min after the injection, a quadrant test in ACET-injected rats showed no significant difference (P > 0.05) in quadrant 4 preference (F3,16 = 132.9, P < 0.0001; Fig. 9C) from that of the saline control rats (F3,16 = 306.4, P < 0.0001; Fig. 9B). These results indicate that once formed, memory and its recall, as well as the sensory stimuli that elicit recall, are not vulnerable to ACET. During the experimental periods, no rats showed any apparent sign of discomfort or abnormal behaviors such as hypo- or hyperactivity.
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DISCUSSION |
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In vitro rhythm and cholinergic involvement
The CCH-induced in our study is consistent with the in vitro
previously reported by many other groups (Golebiewski et al.
1996
; Huerta and Lisman 1993
,
1995
; Konopacki and Golebiewski 1993
;
Pitler and Alger 1992
; Vertes and Kocsis
1997
; Williams and Kauer 1997
) and appears to be
fundamentally identical to the
rhythm in vivo for its sensitivity
to muscarinic receptor antagonists, dependence on GABAergic
interneurons, and independence of glutamatergic inputs.
Acetylcholine's activation of muscarinic receptors on pyramidal cells
is considered to be modulatory and much too slow to generate rhythmic
directly (Dutar et al. 1995
; Vertes and Kocsis 1997
). The ineffectiveness of blocking glutamatergic
inputs on the
is also consistent with the evidence that during
oscillations in vivo CA3 neurons rarely reach action potential
threshold (Bland and Wishaw 1976
; Fox and Ranck
1981
) and excitatory inputs from CA3 are unlikely to contribute
to CA1
(Soltesz and Deschenes 1993
; Thompson
and Best 1989
; Ylinen et al. 1995
). The
effectiveness of the specific GABAA receptor
antagonist BIC in eliminating the postsynaptic response and
activity strongly suggests that GABAB receptor
activation did not contribute significantly to the responses. The CA1
activity does, however, appear to be distinct from the activity
oscillations that were BIC insensitive, involved epileptiform bursting,
and were generated by CA3 neurons in one report (William and
Kauer 1997
). This difference may depend on the preparations or
age of the animals. In their study, slices were obtained from younger
animals so that cells may have a high intracellular
Cl
concentration, due to the lack of a
developmentally expressed Cl
-extruding
K+/Cl
co-transporter in
early age (Rivera et al. 1999
).
Involvement of muscarinic receptors in hippocampal induction has
been well established. Low-frequency MS/DBv stimulation activates
cholinergic inputs to the hippocampus and drives
in vivo
(Descarries et al. 1997
). Microinfusions of CCH or
eserine into areas including CA1 induce an atropine-sensitive
hippocampal
activity in vivo (Rowntree and Bland
1986
). Atropine administration has been found to eliminate
hippocampal
in vivo (Brazhnik and Vinogradova 1986
;
Vertes and Kocsis 1997
). The effectiveness of muscarinic
antagonists does not mean, however, that there is only one form of
.
In anesthetized rats, atropine eliminates
(Stewart and Fox
1989
). Under such conditions, an unconventional small "residual
" was described, that, in the absence of
, could be shown by using the activity of the MS/Dev neurons that discharged rhythmically to trigger hippocampal EEG in analysis (Stewart and Fox 1989
). The involvement of serotonergic transmission has
been proposed (Vanderwolf et al. 1987
). The particular
role and importance of such an atropine-resistant component in memory
remains to be established. Furthermore, intracellular
activity of
pyramidal cells has been claimed to result from depolarizing or
hyperpolarizing membrane potential oscillations (Vertes and
Kocsis 1997
).
The activities induced in our study were most likely evoked by
muscarinic receptor activation, given their sensitivity to atropine. We
do not, however, rule out the possibility that multiple cell targets
might be required for CCH to induce the
. Nor can we rule out the
involvement of the nicotinic receptors entirely. However, it has been
shown that the interneurons in or near the stratum pyramidale and with
axonal projections within and around this layer exhibit no nicotinic
response (McQuiston and Madison 1999
).
HCO3-mediated GABAergic synaptic depolarization
Encoding experiences into lasting memory may involve a qualitative
diversity of synaptic plasticity (Brenowitz et al. 1998; Kornhauser and Greenberg 1997
; Otis et al.
1996
; Paulsen and Moser 1998
), including
changing operations of preexisting synapses and growing new ones.
GABAergic postsynaptic depolarizing responses have been observed by
several groups (Alkon et al. 1992
; Kaila et al.
1993
; Michelson and Wong 1991
; Rivera et
al. 1999
; Siklós et al. 1995
;
Staley et al. 1995
). The depolarization induced in the
present study differs from that reported by Kaila et al.
(1997)
, who applied a high-frequency train of pulses to the
stratum radiatum to induce depolarizing responses that showed a slow
time course but lasted for several seconds. Nevertheless, our results
are consistent with the evidence that GABAergic depolarization can be
induced by enhancing HCO3
conductance through
GABAA receptor channels in adult hippocampal cells, a response sensitive to carbonic anhydrase inhibitors
(Kaila et al. 1993
; Staley et al. 1995
).
Carbonic anhydrase exists in pyramidal cells (Pasternack et
al. 1993
). Indeed, the
activity and the reversed GABAergic
postsynaptic responses were largely abolished by carbonic anhydrase
inhibitors. The effectiveness of intracellular benzolamide, a
membrane-impermeable carbonic anhydrase inhibitor, indicates that the
response depends on activity of an intracellular enzyme. Supporting the
functional importance of carbonic anhydrase activity in synaptic
plasticity is also the result that a partial blockade of the enzyme
activity in vivo markedly impaired retention of rat watermaze learning.
HCO3
has a reversal potential about
12 mV
(Staley et al. 1995
). With an increased
HCO3
/Cl
permeability ratio,
outward HCO3
flux would depolarize the membrane at
the resting membrane potential. Alteration in HCO3
conductance and/or transmembrane concentrations (thus the driving force) would be expected to dramatically alter the synaptic response.
Regulation of carbonic anhydrase activity is not an entirely new
concept. The existence of physiological regulators of carbonic anhydrase has been proposed, including those that activate the anhydrase by facilitating its membrane association (Parkes and Coleman 1989). Activation of cholinergic receptors is well
known to increase cytosolic Ca2+ concentrations,
at least partially through Ca2+ release from
intracellular stores (Seymour-Laurent and Barish 1995
).
Enhancing effects by Ca2+ on the anhydrase
activity was suggested by the calexcitin and ryanodine receptor results
(Sun et al. 1999
), although the identity of the
intracellular signaling intermediate(s) remains to be determined. In
molluscan neurons, the carbonic anhydrase-HCO3
system has been found to be the most potent regulatory factor in
intracellular pH regulation. Depolarized snail neurons, for example,
were associated with increased proton conductance (e.g., Thomas
and Meech 1982
). Changes in intracellular pH could also alter
ion channel function as well as metabolic activity. It remains to be
determined whether intracellular pH is significantly altered or plays a
role in the CCH-induced
and/or regulation of memory behavior.
Phase relationship of activities of CA1 pyramidal cells and
interneurons
Two major classes of hippocampal CA1 neurons are the cells and
the "place cells" (Paulsen and Moser 1998
). The
GABAergic interneurons, including basket cells and axo-axonic cells,
have been called
cells (Paulsen and Moser 1998
). The
basket interneurons are particularly active and express strongest
rhythmic discharges (Cscsvari et al. 1999
) when
hippocampal EEG is dominated by
rhythm. One basket interneuron
selectively and perisomatically innervates approximately 1,000 pyramidal cells (Cobb et al. 1995
) and thus can entrain
a large population. The pyramidal neurons, on the other hand, are
largely quiescent during the
rhythm associated with exploration,
but a subpopulation shows strong firing that is highly correlated with
specific locations in space (Dutar et al. 1995
;
Paulsen and Moser 1998
; Vertes and Kocsis
1997
). These "place" cells fire at all phases of the
rhythm (O'Keefe and Recce 1993
). Our results show that
during
oscillation, the GABAergic postsynaptic responses are
altered. Gating through a postsynaptic mechanism, as described in the
present study, could explain why some pyramidal cells become active
while the vast majority of others remain silent during
EEG, even if
they are innervated by the same interneuron.
Every pyramidal cell is innervated by 10-12 GABAergic interneurons,
preferentially making synapses on cell bodies, proximal dendrites, and
axon initial segments of CA1 pyramidal cells (Buhl et al.
1994; also Paulsen and Moser 1998
for review).
If pyramidal cells were activated by rebound excitation from GABAergic
inhibition, one would expect that pyramidal cells should discharge
when interneurons become silent. This may be the case in anesthetized
states. The intracellular
activity of CA1 pyramidal cells when
recorded under anesthesia have often been reported to fire
out-of-phase, delayed about a half cycle (Soltesz and Deschenes
1993
; Ylinen et al. 1995
). In behaving animals
or during REM sleep, however, the earlier discharge peaks of these
interneurons precede peaks of population activity of pyramidal cells
during
activity, and both pyramidal cell firing and interneuronal
discharge occur within the same
phase period (Cscsvari et
al. 1999
). Anesthesia is known to attenuate a large peak of
, revealing rhythmic hyperpolarization of pyramidal cells from
basket interneurons (Ylinen et al. 1995
). Thus the
activity during exploration or induced by cholinergic agonists in vitro
seems not directly comparable to the
under anesthesia (Muir
and Bilkey 1998
; Ylinen et al. 1995
).
Not only does the discharge phase relationship between pyramidal cells
and interneurons differ between the anesthetized and behaving animals,
but the phase relationship is also dynamic. In behaving animals, the
phase forward shift of the discharges of place cells on each cycle
occurs during traversal of the place field of the cell (O'Keefe
and Recce 1993
; Shen et al. 1997
). Place cells
thus fire in phase with progressively stronger GABAergic inputs from
interneurons and at earlier phases of the
cycles as the rat moves
toward the center of their place field (Fig. 7B)
(Csicsvari et al. 1999
; O'Keefe and Recce
1993
; Shen et al. 1997
). Mechanism(s)
responsible for the
initiation or entraining of the pyramidal cell
activity should be able to code for timing (Trussell
1999
) or entrain pyramidal cells at different
phases, including those with minimal delay (Cscsvari et al.
1999
). A rebound excitation following hyperpolarization is
unlikely to have such multi-phase capability as the interneurons become
more active during
-related activity. A brief switch toward or to
GABAergic depolarization would be more effective and reliable in
processing dynamic information. Strong GABAergic inputs after the
synaptic switch can entrain the activity of pyramidal cells so that the delay would be relatively short and evoke an "in phase" activity.
The present results suggest that the GABAergic entraining could result
through in three ways. In a small percentage of cells, CCH was able to
elevate the reversal potential to levels that were above the threshold
for spike activity. GABAergic inputs thus could directly drive, even if
briefly, activity of the pyramidal cells with sufficient transformation
of hyperpolarizing to depolarizing responses. The reversed response,
although often not strong enough to reach threshold by itself, can
entrain the pyramidal cells when stimulated at a frequency (Fig.
7). Furthermore, the reversed response can effectively enhance weak
excitatory inputs to reach threshold (Fig. 7C) (Sun
et al. 1999
). When the inputs are not very strong and require
summation of multiple synaptic activation or other associative inputs,
such as glutamatergic inputs (Sun et al. 1999
) to reach
the threshold, the entrained action potentials are likely to be
delayed. It should also be noted that for this summation effect to
occur, there must be sufficient spatial proximity on the pyramidal
cells for the glutamatergic excitatory postsynaptic potentials to
spread from the dendrites to the somata where they would interact with
the transformed GABAergic IPSPs. Thus place cells would be capable of
firing at all phases of the
rhythm in relation to the activity of
GABAergic interneurons.
We have shown reversed,
HCO3-dependent GABAergic postsynaptic
responses and their effectiveness in entraining activity of pyramidal
cells. The most reasonable explanation for our results is an essential
requirement of carbonic anhydrase activity in the molecular signaling
pathways for learning and memory. This explanation is consistent with
the occurrence of mental retardation in carbonic anhydrase
II-deficient patients (Sly and Hu 1995
). Carbonic
anhydrase is very efficient and may act as a functional switch. The
effectiveness of its inhibition on impairing rat spatial memory does
not necessarily mean, however, that the bicarbonate-dependent GABAergic
depolarization, as defined in vitro, also directly contributes to
spatial memory. The critical role of HCO3
in
activity is also consistent with the fact that ACET is effective in the treatment of central sleep apnea or epilepsy, causing somnolence together with significant decreases in centrally originated
rhythm-related fluctuations in cardiorespiratory functions
(Sone et al. 1998
). ACET-regulated
HCO3
gradients appear important for acquisition of
memory rather than retrieval from formed memory. Such compounds may
have clinical value when temporarily suppressed memory is beneficial
(e.g., in surgery or posttraumatic stress disorder).
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ACKNOWLEDGMENTS |
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We thank Dr. T. H. Maren for kindly providing benzolamide.
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FOOTNOTES |
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Address for reprint requests: M.-K. Sun, Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke/NIH, Bldg. 36, Rm. 4A24, 36 Convent Dr., Bethesda, MD 20892 (E-mail: mksun{at}codon.nih.gov).
Received 14 February 2000; accepted in final form 21 September 2000.
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REFERENCES |
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