Department of Neurophysiology, Division of Neuroscience, The Medical School, The University of Birmingham, Birmingham B15 2TT, United Kingdom
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ABSTRACT |
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Bracci, Enrico, Martin Vreugdenhil, Stephen P. Hack, and John G. R. Jefferys. Dynamic Modulation of Excitation and Inhibition During Stimulation at Gamma and Beta Frequencies in the CA1 Hippocampal Region. J. Neurophysiol. 85: 2412-2422, 2001. Fast oscillations at gamma and beta frequency are relevant to cognition. During this activity, excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) are generated rhythmically and synchronously and are thought to play an essential role in pacing the oscillations. The dynamic changes occurring to excitatory and inhibitory synaptic events during repetitive activation of synapses are therefore relevant to fast oscillations. To cast light on this issue in the CA1 region of the hippocampal slice, we used a train of stimuli, to the pyramidal layer, comprising 1 s at 40 Hz followed by 2-3 s at 10 Hz, to mimic the frequency pattern observed during fast oscillations. Whole cell current-clamp recordings from CA1 pyramidal neurons revealed that individual stimuli at 40 Hz produced EPSPs riding on a slow biphasic hyperpolarizing-depolarizing waveform. EPSP amplitude initially increased; it then decreased concomitantly with the slow depolarization and with a large reduction in membrane resistance. During the subsequent 10-Hz train: the cells repolarized, EPSP amplitude and duration increased to above control, and no IPSPs were detected. In the presence of GABAA receptor antagonists, the slow depolarization was blocked, and EPSPs of constant amplitude were generated by 10-Hz stimuli. Altering pyramidal cell membrane potential affected the time course of the slow depolarization, with the peak being reached earlier at more negative potentials. Glial recordings revealed that the trains were associated with extracellular potassium accumulation, but the time course of this event was slower than the neuronal depolarization. Numerical simulations showed that intracellular chloride accumulation (due to massive GABAergic activation) can account for these observations. We conclude that synchronous activation of inhibitory synapses at gamma frequency causes a rapid chloride accumulation in pyramidal neurons, decreasing the efficacy of inhibitory potentials. The resulting transient disinhibition of the local network leads to a short-lasting facilitation of polysynaptic EPSPs. These results set constraints on the role that synchronous, rhythmic IPSPs may play in pacing oscillations at gamma frequency in the CA1 hippocampal region.
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INTRODUCTION |
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The hippocampal slice provides
several models for the study of fast oscillations. In particular, CA1
neurons can generate rhythmic activity at gamma (30-100 Hz) and beta
(10-30 Hz) frequencies following chemical or electric stimulation
(Bracci et al. 1999; Fisahn et al. 1998
;
Whittington et al. 1995
, 1997a
). Mathematical models of
this activity (Traub et al. 1996a
,b
; Wang and
Buzsáki 1996
; Whittington et al. 1997a
)
are based on the generation of rhythmic, synchronous synaptic
potentials, which are thought to play an essential role in timing
collective neuronal firing. These models have not yet included
realistic activity-dependent, short-term modifications of synaptic
events (Arai and Lynch 1996
; Davies and
Collingridge 1993
, 1996
; Davies et al. 1990
).
However, these modifications are very likely to take place and might
affect dramatically synaptic communication during fast oscillations.
The picture is complex, involving at least four different receptor
classes [N-methyl-D-aspartate (NMDA), AMPA,
GABAA, and GABAB]. The
use-dependent changes of responses mediated by each of these receptors
have been investigated (Arai and Lynch 1996
;
Davies and Collingridge 1993
, 1996
; Davies et al.
1990
; Debanne et al. 1996
), commonly in
pharmacological isolation and with low-intensity stimuli delivered in
pairs or at fixed frequencies. However, the way in which glutamatergic and GABAergic responses interact during synchronous oscillatory activity is difficult to predict from these data. Summation of postsynaptic potentials can be highly nonlinear due to factors such as
shunting effects on synapses at particular electrotonic locations
(Andersen et al. 1980b
; Harris et al.
1992
) and activation of presynaptic receptors (Cobb et
al. 1999
; Cohen et al. 1992
; Colmers et
al. 1988
; Davies and Collingridge 1993
;
Davies et al. 1990
). Furthermore, even for a single
receptor class, strong nonlinear summation of responses may arise from
receptor saturation (Liu et al. 1999
),
concentration-dependent rate of transmitter clearance from synaptic
cleft (Clements 1996
; Otis et al. 1996
;
Roepstorff and Lambert 1994
; Scanziani et al.
1997
), activation of extrasynaptic receptors (Pham et
al. 1998
; Rusakov and Kullmann 1998
), and
changes in transmembrane ionic concentration gradients
(Schwartzkroin et al. 1998
; Staley et al.
1995
).
A practicable experimental approach consists in studying a particular
pattern of synaptic activation that is specifically relevant to a
certain phenomenon, as done by Davies and Collingridge (1996) to investigate the interaction between GABA and
glutamate receptors during priming protocols that enhance LTP
expression in the CA1 region.
During fast oscillations in vitro, a large number of CA1 pyramidal
neurons and interneurons fire simultaneously on each cycle (Traub et al. 1996b; Whittington et al.
1997a
). Therefore CA1 neurons receive rhythmic, simultaneous
inhibitory and excitatory inputs from many other presynaptic CA1
neurons. To cast light on the dynamic changes accompanying this
repetitive synaptic activation, we used a protocol comprising a train
of stimuli at 40 Hz followed by one at 10 Hz to mimic the frequency
pattern observed (Bracci et al. 1999
; Traub et
al. 1999
; Whittington et al. 1997b
). Stimulation was applied in the CA1 region to activate local pyramidal neurons and
interneurons. The responses evoked by this experimental protocol were
not dependent on cholinergic or metabotropic glutamate receptors (as
shown by application of specific antagonists), thus allowing a
dissection of the effects of GABAergic and ionotropic glutamatergic events from those due to slower changes of membrane properties. Activation at gamma and beta frequency was found to produce a profound
short-term alteration of the balance between excitation and inhibition
within the local network.
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METHODS |
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Transverse hippocampal slices (400 µm), were prepared from young male Sprague-Dawley rats (90-120 g; anesthetized with ketamine and medetomidine). Slices were maintained (and experiments were performed) at 32-36°C in gassed (5% CO2-95% O2) artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 26 NaHCO3, 2 CaCl2, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, and 10 glucose. Whole cell recordings were obtained from visualized pyramidal neurons; slices were submerged in a chamber mounted on an Olympus XY212 upright microscope with a ×40 water-immersion objective and differential interference contrast optics. Patch pipettes contained (in mM) 145 potassium-gluconate, 10 KCl, 10 HEPES, 2 NaCl, 2 Mg-ATP, and 0.1 EGTA, pH = 7.25 (adjusted with KOH). GTP was omitted to minimize the effects of G-protein-dependent processes. A stimulating electrode (consisting of a glass pipette filled with ACSF) was placed in the stratum pyramidale of the CA1 region. Tight-seal whole cell recordings were obtained from visualized neurons located within 50 µm from the stimulation site. The stimulation protocol consisted of two consecutive trains, one at 40 Hz (for 1 s) followed by another at 10 Hz (for 2-3 s). Pulse duration was 0.1 ms. A single stimulus was usually delivered 1 s before and 1 s after this protocol to monitor the changes in the evoked response.
Modulation of the evoked postsynaptic responses, and of the associated slow depolarization (see RESULTS), were observed when the stimulation intensity was sufficiently strong. The stimulation intensity was routinely set at 1.5 times the minimum required to elicit a slow depolarizing response during the 40/10-Hz stimulation (defined as threshold; 10-30 V, 0.1-ms duration).
The protocol was repeated every 5 min to allow a complete recovery to control conditions. Whole cell patch-clamp recordings were made using an Axoclamp 2B amplifier under current-clamp conditions in bridge mode. Data were stored and analyzed using SIGNAL software (CED, Cambridge, UK). Statistical significance was assessed by one-way ANOVA (Sigmaplot, SPSS). Data are expressed as means ± SD.
Staley and Proctor (1999) have shown that depolarizing
GABA responses mainly arise from dendrites, where surface to volume ratio is larger than in the soma. These authors used a standard electric circuit model of the dendrite to simulate chloride
accumulation taking place after focal GABA application or tetanic
stimulation. We used a similar model to simulate the voltage-dependent
behavior observed in the present study. The model included the
following features: membrane potential was determined by the
equilibrium condition of the currents flowing through a set of
conductive pathways (each characterized by its reversal potential
determined by the correspondent ionic gradient) and the externally
injected current; the GABAA receptor mediated
conductance consisted of two different electrochemical pathways,
representing the bicarbonate permeability and the chloride permeability
of these receptors, respectively; conductance ratio of these pathways
was fixed and equal to 0.25; intracellular chloride concentration (and
thus chloride reversal potential calculated through the Goldman
equation) was dynamically determined by chloride flow-through GABA
conductances, by chloride extrusion through a potassium-chloride
cotransporter and by a constant chloride leak into the cell; transport
rate (v) of the cotransporter was determined by the
Lineweaver-Burke equation as a function of intracellular chloride
concentration [Cl]i
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RESULTS |
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Single shock stimulation delivered to the pyramidal layer gave
usually rise to a typical EPSP-IPSP sequence in pyramidal neurons (current-clamped between 60 and
55 mV, close to their physiological resting potential; Fig. 1A).
The evoked IPSP was mainly mediated by GABAA
receptors with little or no contribution from
GABAB receptors, as indicated by its
monoexponential decay time course and its insensitivity to application
of the GABAB antagonist CGP 55845A (1 µM; see
Fig. 6). This was due, at least in part, to GTP-free intracellular
solution, used here to minimize metabotropic receptor influence on the
postsynaptic cell (Pham et al. 1998
; Saugstad et
al. 1998
). In a small number of cells (n = 4),
it was possible to elicit a purely hyperpolarizing response with a
single stimulus delivered to the stratum pyramidale (Fig.
1B). During 40-Hz stimulation, IPSPs fused together and
could not be detected individually as also was apparent in the presence
of ionotropic glutamate receptor blockers (see Fig. 7). The signals
observed during the 40/10-Hz protocols are therefore better described
in terms of the evoked EPSPs, which remained individually discernible,
and of a slower polarizing waveform on which these EPSPs were
superimposed. Provided the stimulation intensity was strong enough,
this slow waveform comprised: an initial hyperpolarization resulting
from fused IPSPs during the early phase of 40-Hz stimulation and a
subsequent depolarization (developing either during the late phase of
40-Hz stimulation, as in the example of Fig. 1A, or during
the early phase of 10-Hz stimulation, as in the example of Fig.
1B). The minimum stimulation intensity able to elicit a slow
depolarization during the 40/10-Hz protocol was defined as threshold
(T, see METHODS). In 55 of 87 neurons tested, a
late hyperpolarization was also present, starting either during the
late phase of 10-Hz stimulation or just after it and persisting for
3-10 s after the end of the stimulation protocol (as in the examples
of Fig. 1, A and B). On average, the amplitude of
these responses (for cells kept between
55 and
60 mV) was 9 ± 5 mV for the early hyperpolarization, 12 ± 6 mV for the slow
depolarization, and 5 ± 4 mV for the late hyperpolarization (calculated only for the group of cells in which such hyperpolarization was observed). When pyramidal neurons were hyperpolarized by current injection to levels more negative than the GABAA
IPSP reversal potential (
77 ± 5 mV, n = 12),
the early hyperpolarization was converted into a depolarization while
the late hyperpolarization (if present) was depressed or abolished.
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The EPSPs elicited by the second to fourth pulses of 40-Hz stimulation
(during the early hyperpolarizing phase of the envelope) were larger in
amplitude than those elicited by a single pulse (by 29 ± 8%,
P < 0.001). This observation cannot be explained solely by the increased driving force for EPSP during the early hyperpolarization because in some cases, the EPSP elicited by the
second to fourth pulses at 40 Hz attained a more positive peak than the
one elicited by a single pulse (not shown); EPSP half-width during the
first eight stimuli at 40 Hz was not significantly different from
single-stimulus response, showing that each response was still
curtailed by an IPSP. These data are consistent with the observations
of Davies and Collingridge (1996), who reported paired-pulse potentiation of AMPA receptor-mediated EPSPs with 20-ms
interval (50 Hz) and paired-pulse depression of <40% for GABAA IPSPs with 25-ms interval (40 Hz)
In the cells in which a pure IPSP was evoked by a single stimulus, the 40/10-Hz protocol still elicited a slow waveform similar to the one observed in the other cases, and EPSPs appeared during the 40-Hz train, starting from the second stimulus (Fig. 1Bii).
In both groups of cells, the amplitude of the evoked EPSPs progressively decreased from the peak amplitude reached by the third to fifth stimulus at 40 Hz. The amplitude of the EPSPs evoked by the 30th-40th stimuli at 40 Hz was on average 21 ± 19% of the maximal one, elicited by the 2nd-4th stimuli (P < 0.001). This decrease could not be attributed to concomitant changes in membrane potential as is clear from comparison of responses elicited at similar potentials but at different times during the early hyperpolarization (Fig. 1, Aii and Bii). On the other hand, the half-width of EPSPs evoked by 30th-40th stimuli at 40 Hz was slightly (12 ± 7%), but significantly (P < 0.05), larger than for a single-stimulus response.
During 10-Hz stimulation, the amplitude and the half-width of the EPSPs
gradually increased to levels well above those observed with a single
pulse or during 40-Hz stimulation. Hyperpolarizing IPSPs were not
detectable during the 10-Hz train. The EPSP potentiation (and the
absence of IPSPs) persisted for several seconds after the 40/10-Hz
protocol; a single stimulus delivered 1 s after the protocol
elicited a monophasic depolarizing response, whose amplitude, duration
and time-to-peak were significantly (P < 0.001) larger than those of the largest EPSPs observed during 40-Hz stimulation (Fig.
2, A and B).
Furthermore, a multi-peak shape was often observed in the responses
elicited during the 10-Hz protocol or 1 s after it (Fig.
2A). These data suggest that the gradual increase of EPSP
during 10-Hz stimulation was due to progressive recruitment of
previously silent polysynaptic pathways. The large (>300%) increase
in EPSP half-width at the end of 10-Hz stimulation could not be merely
attributed to the absence of a curtailing IPSP because this phenomenon
is expected to produce an increase in half-width <90% even for >70%
depression of the IPSP (Davies and Collingridge 1996).
No significant differences were found in EPSP modulation between cells
in which the late hyperpolarization was present and those in which this
event was absent.
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In 34% of cells tested, facilitated EPSPs elicited after the 40/10 Hz were large enough to generate action potentials that were not present with the same stimulation before the 40/10-Hz protocol (Fig. 3). Thus modulation of the evoked postsynaptic potentials is physiologically relevant being able to convert subthreshold signals into superthreshold ones.
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When monitored with 1-Hz stimulation, evoked PSPs recovered to their prestimulation shape (comprising an EPSP-IPSP sequence) within 30-45 s from the end of the 40/10-Hz protocol. More persistent changes in the EPSP amplitude were not observed, suggesting that the present protocol did not cause long term potentiation of the evoked responses.
These data show that at the end of the 40/10-Hz protocol the local network was strongly biased toward excitation (apparent in the vast increase of polysynaptic EPSPs and in the absence of hyperpolarizing IPSPs). One of the factors affecting postsynaptic responses is the membrane input resistance, therefore we tested the changes induced by 1 s of 40-Hz stimulation. As shown in Fig. 4, membrane resistance collapsed to ~20% at the end of 40-Hz stimulation, i.e., when a large reduction in EPSP amplitude was observed (on average, by 77 ± 11%). Membrane resistance gradually recovered to control value either in the absence of stimuli (Fig. 4) or during 10-Hz stimulation (not shown). Recovery to 90% of control value was complete within 1.6 ± 0.6 s from the end of 40-Hz stimulation (n = 4) in the absence of stimuli. This drop in input resistance can account for the decrease in EPSP amplitude observed during the late part of the 40-Hz train and the first part of the 10-Hz train.
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The temporal link between the changes in EPSPs and the different phases
of the slow polarizing waveform led us to suspect that the cellular
mechanisms responsible for the generation of such a slow signal could
also be involved in EPSP modulation. The slow waveform and the evoked
potentials were not affected by the metabotropic glutamate receptor
(mGluR) antagonist MCPG (1 mM; n = 5) or by the
muscarinic acetylcholine receptor antagonist atropine (1 µM;
n = 4; data not shown). Slow
hyperpolarizing-depolarizing waveforms are typical of responses to the
sustained presence of GABA, which can be elicited in CA1 pyramidal
neurons by several protocols, including high-frequency stimulation
(Bracci et al. 1999; Kaila et al. 1997
;
Taira et al. 1997
) and focal GABA application (Staley et al. 1995
). To check whether this kind of
effect was involved under the present conditions, we applied selective
GABAA receptor antagonists, which are known to
block depolarizing GABA effects (Kaila et al. 1997
;
Staley et al. 1995
). Bath application of either
bicuculline (50 µM) or picrotoxin (100 µM) caused major changes in
the slow waveform observed during the 40/10-Hz protocol as illustrated
in Fig. 5A. In the presence of
either of these agents, the early hyperpolarization was converted into
a depolarizing burst which persisted as long as 40-Hz stimuli were
delivered (n = 7); when the stimulation frequency
switched to 10 Hz, a rapid repolarization to baseline, followed by a
hyperpolarization [taking place either during (Fig. 5C) or
after 10-Hz stimulation (Fig. 5A)], was observed. These
changes in the slow waveform were accompanied by major changes in the
evoked EPSPs. During 10-Hz stimulation, each stimulus elicited an
individually discernible EPSP but, unlike in control solution, the
amplitude and duration of these responses were already maximal at the
beginning of the 10-Hz train and did not increase progressively (as
quantified in Fig. 5B). When, in the presence of
GABAA blockers, responses elicited by a single pulse before the protocol or 1 s after it were compared no
difference in half-width was detected, while the amplitude of the
postprotocol EPSP was slightly but significantly reduced (by 16 ± 6%, P < 0.001). In other words, the duration of EPSPs
evoked in control solution after the stimulation protocol was similar
to the one observed with a single pulse in the presence of bicuculline
or picrotoxin, and in the presence of these agents, no further
broadening of evoked EPSPs could be induced by the stimulation trains.
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These data suggest that GABAA receptor-mediated
effects were mainly responsible for the slow envelope and for the
dynamic changes in the evoked EPSPs. Since GABA release is controlled by presynaptic GABAB receptor (Davies and
Collingridge 1993), we tested the effects of a selective
antagonist of this receptor. In the presence of CGP 55845A (1 µM,
n = 4), the induction of the slow depolarizing waveform
and the associated modulation of EPSPs was strongly facilitated so that
the minimum stimulation intensity required to elicit a slow
depolarization during the 40/10-Hz protocol was considerably reduced
(by 57 ± 21%; P < 0.001). Thus low stimulation
intensities, which elicited no depolarizing responses and gave rise to
little and inconsistent dynamic modulation of EPSPs in control
solution, were sufficient to elicit a slow depolarization in the
presence of CGP 55845A as illustrated in the example of Fig.
6. If the slow waveform ended in a late
hyperpolarization in control solution, this was not abolished by CGP
55845A (Fig. 6). The use of whole cell recordings without intracellular
GTP virtually abolished the effect of postsynaptic
GABAB receptor activation (as mentioned in the
preceding text). We conclude that other cellular mechanisms must be
responsible for the observed afterhyperpolarization but did not
investigate this further because the afterhyperpolarization did not
appear to be linked to EPSP modulation. When stimulation intensity was
adequately reduced in the presence of CGP 55845A, EPSP modulation
during the 40/10-Hz protocol was in all respects similar to those
observed in control solution with stronger stimulation intensities (see
METHODS for a description of the intensities used). On the
other hand, when the stimulation intensity was similar to that
previously used in control solution, the 40/10-Hz protocol elicited a
paroxysmal slow depolarization associated with intense firing activity,
which often prevented the identification of individual evoked EPSPs (Fig. 6C). Under these conditions, a single shock applied
1 s after the end of the 40/10-Hz protocol elicited a large EPSP
accompanied by a burst of two to four action potentials (Fig.
6C). These results are consistent with recent reports on the
role of presynaptic GABAB receptor in modulating
depolarizing GABA effects (Manuel and Davies 1998
). In
the presence of GABAB antagonist, the
negative-feedback of GABA release is impaired and much more GABA is
released for the same stimulation (Cobb et al. 1999
). As
a consequence, depolarizing GABA effects, which require large levels of
transmitter release to be expressed (Staley et al.
1995
), are strongly facilitated.
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To gain further insight into the cellular mechanisms mediated by
GABAA receptors during 40/10-Hz activation, we
performed experiments in which the other transmitters involved in this
phenomenon were pharmacologically blocked. This was accomplished by
simultaneous bath-application of the AMPA receptor antagonist
2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX)
(50 µM), the NMDA receptor antagonist D-AP5 (50 µM),
and the GABAB antagonist CGP 55845A (1 µM). In
the presence of these agents, polysynaptic IPSPs are blocked and fewer
GABAergic terminals are activated by a given stimulation intensity; on
the other hand, as described in the preceding text, GABA release from
terminals activated directly by the stimulus is facilitated by block of GABAB presynaptic receptors (Bracci et al.
1999; Davies and Collingridge 1993
). Under these
conditions, it was possible to adjust the stimulation intensity to
evoke a slow hyperpolarizing-depolarizing waveform of similar amplitude
to that in control solution. This was accomplished by changing the
stimulation intensity by a factor of 0.7-2.5 with respect to control
solution. In the presence of these agents, the slow depolarization was
accompanied by a decrease in input resistance similar in extent and
time course to that observed in control solution (not shown), showing
that such a decrease was largely due to activation of
GABAA conductances.
The cellular mechanisms of depolarizing GABA effects are controversial.
Intradendritic chloride accumulation (Staley and Proctor 1999; Staley et al. 1995
) and extracellular
potassium accumulation (Kaila et al. 1997
) appear to
play different roles under different experimental conditions. We
performed experiments to identify the main mechanism operating under
the present conditions. Extracellular potassium accumulation is
produced by the local CA1 network following tetanic stimulation at 100 Hz (Kaila et al. 1997
). The time course of this
phenomenon should not be strongly affected by manipulations of the
membrane potential of the recorded neuron. On the other hand,
transmembrane chloride movements are expected to depend on the membrane
potential of the individual neuron. Whole cell recording technique
allowed us to manipulate the membrane potential of the current-clamped
cell reliably over a large range of values while the 40/10-Hz protocol
was applied. Typical results of these experiments are shown in Fig.
7. When the cell was current-clamped at
50 mV, a single shock elicited a hyperpolarizing IPSP; a
hyperpolarizing-depolarizing sequence was observed during the 40/10-Hz
protocol, as in control solution. Phasic responses were markedly
attenuated or absent during the 10-Hz train; a single stimulus applied
1 s after the end of 10-Hz train (at a membrane potential similar
to the preprotocol one) elicited little or no response. When the neuron
was held at
95 mV, a single stimulus evoked a depolarizing IPSP.
During the 40-Hz train, the cell underwent a brisk depolarization
resulting from summed depolarizing IPSPs, followed by a slower one,
which gradually recovered to baseline during the 10-Hz train. Phasic depolarizing IPSPs were absent during the first 3-10 stimuli at 10 Hz
but were clearly detectable with the subsequent stimuli. A single
stimulus applied 1 s after the end of the 10-Hz train elicited a
depolarizing IPSP characterized by smaller amplitude (on average by
29 ± 19%, n = 4, P < 0.001)
than the one elicited before the 40/10-Hz protocol. An important
feature of this phenomenon was that in all the cells tested
(n = 5), the slow depolarization reached its peak value
at different times under the two conditions. The peak was attained
significantly (P < 0.001) later (670 ± 210 ms
after the end of 40-Hz train) when the cell was depolarized (between
40 and
50 mV) than when it was hyperpolarized (between
90 and
100 mV; peak 47 ± 67 ms after the end of 40-Hz train). When the
cell was kept close to IPSP reversal potential, 40-Hz stimulation
evoked a slow monophasic depolarization that persisted during the early
phase of 10-Hz stimulation and then progressively declined to baseline
(Fig. 7B); during the late phase of 10-Hz stimulation,
phasic depolarizing IPSPs were apparent. Similarly, a single stimulus
delivered after 1 s after the 10-Hz train elicited a clearly
discernible depolarizing response (Fig. 7B). These findings suggested that major changes in GABAA receptor
driving force occurred during the 40/10-Hz protocol and that the
modifications were sensitive to the state of polarization of the neuron
membrane. The observation that the kinetics of the slow waveform
depended on the membrane potential of the recorded cell suggested that
a network-mediated extracellular accumulation of potassium was not the
major factor responsible for these events.
|
To acquire more direct information on potassium accumulation, we
performed whole cell recordings from glial cells (n = 3) located in the pyramidal layer during the 40/10-Hz protocol. A typical example of these experiments is shown in Fig.
8. When stimulation intensity was at
threshold for the observation of the slow potential envelope (and the
associated modulation of EPSPs) in local pyramidal neurons, the glial
cell (kept without injected current at 68 mV) displayed a 4-mV
depolarization during the 40-Hz train. A further small depolarization
(<1 mV) was observed during the 10-Hz train, followed by a slow
repolarization to baseline, which took several seconds. When the
stimulation intensity was increased to 1.5 times T, a larger
depolarization was elicited by the 40-Hz train, and the cell continued
to depolarize (by >2 mV) during the 10-Hz train. The time course of
this behavior was clearly different from that observed in an adjacent
pyramidal neuron (and typical of this kind of cell). In this cell, the
depolarization observed during the 40-Hz train was followed (both with
1 or 1.5 times T stimulation) by an immediate repolarization
during the 10-Hz train. Furthermore, despite the expected greater
sensitivity of glial cells to increases in extracellular potassium
(Lothman and Somjen 1975
), the depolarization observed
in the neuron during the 40-Hz train was more than double that in the
glial cell. These results suggested that the 40/10-Hz protocol did
cause a local increase in extracellular potassium, but this phenomenon
could not be considered as the main cause of the slow depolarization observed in pyramidal neurons.
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To test whether the experimental observations could be explained in
terms of dendritic intracellular chloride accumulation, we performed
numerical simulations using a standard electrical model similar to the
one used by Staley and Proctor (1999) (Fig. 9A; see also
METHODS). The results of the simulations are shown in Fig.
9, B-F. Reversal potential for chloride
(ECl) and for GABAA receptor
(EGABA, depending on chloride and
bicarbonate transmembrane gradients through the Goldman-Hodgkin-Katz
equation) and membrane potential (Em)
are plotted versus time (Fig. 9, B-D). The time course of
GABAA conductance
(gGABA) (same for all simulations) is
shown in Fig. 9F. Constant amounts of positive or negative current were injected in the model dendrite to reproduce the
experimental conditions of Fig. 7.
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When current injection was such that at rest
Em = EGABA (Fig. 9B),
intracellular chloride accumulation caused a depolarizing shift of
EGABA. In agreement with experimental
observations (see Fig. 7B), this shift was such that
EGABA remained more positive than
Em for several s after closure of
gGABA. When the dendrite was kept at
50 mV by positive current injection (Fig. 9C), a hyperpolarizing-depolarizing sequence was present with the slow depolarization peaking at 1,670 ms. When the cell was hyperpolarized to
95 mV (Fig. 9D), a slow, monophasic depolarization was
present. This depolarization peaked much earlier (at 1,185 ms) than the one observed with positive current injection. The cause of this difference in time course can be understood as follows. When the cell
was depolarized by current injection, the initial driving force for
chloride was >60 mV and caused a massive chloride influx during
gGABA activation. This resulted in a
large increase in intracellular chloride (plotted in Fig.
9E) and a consequent depolarizing shift of
ECl and
EGABA. As expected from
thermodynamical considerations, ECl
remained more negative than Em during
the period in which gGABA increased
(Perkins and Wong 1997
). However,
EGABA became more positive than
Em after ~750 ms. At the time when
gGABA peaked, Em
ECl = 2.7 mV and a substantial
chloride influx persisted for 1 s after this peak. Thus intracellular
chloride continued to increase during this period (Fig. 9E)
and ECl and
EGABA continued to depolarize (Fig.
9C). As a result, despite
gGABA decreasing, the product
gGABA × EGABA (which is the variable term in
the expression for Em) continued to
rise for several hundreds of ms after
gGABA peak. Thus the cell continued to
depolarize despite the ongoing decrease of
gGABA. On the other hand, at
hyperpolarized potentials internal chloride accumulation was much more
limited (Fig. 9E) because the initial driving force for
chloride was much smaller (~15 mV). At the time when
gGABA peaked, the driving force for chloride was <1 mV. Thus the rate of chloride accumulation was too
small for the increase in EGABA to
counterbalance the decrease in gGABA.
As a consequence, Em soon started to
decrease, giving rise to an earlier repolarization (and an earlier
recovery of the intracellular chloride transient). It should be noted
that the results of these simulations depend on the characteristics of
the potassium-chloride cotransporter (see METHODS). In
particular, if the maximum pumping rate
vmax were much larger than 5 mM/s, a
much faster clearance of intracellular chloride (and therefore a much
faster recovery of ECl) would be seen
when the neuron was depolarized.
These data show that intracellular chloride accumulation can account
for the differences in the slow depolarization kinetics observed at
different membrane potentials. The simulations also account for the
time course of the driving force for GABAA
receptor mediated potentials (i.e., Em EGABA) observed experimentally with
different levels of membrane polarization; when the cell is depolarized
(Fig. 9C), this driving force (which is >30 mV at rest)
reverses during the rising phase of the slow depolarization, and
persists at levels <5 mV (in absolute value) for several s after the
peak of the slow depolarization. Such a small value can account for the
persistent absence of detectable postsynaptic potentials observed
experimentally during 10-Hz stimuli and with a single stimulus
delivered 1 s after the end of the 40/10-Hz protocol during block
of fast glutamatergic transmission (Fig. 7A). On the other
hand, when the cell is hyperpolarized the driving force
Em
EGABA (which remains negative all the
time) becomes >10 mV (in absolute value) soon after the depolarization
peak. This observation thus explains the earlier reappearance of
depolarizing IPSPs observed experimentally when negative current is
injected into the cell (Fig. 7A).
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DISCUSSION |
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In this study, we used stimulation of the pyramidal layer at gamma
and beta frequency to mimic the pattern of activation of excitatory and
inhibitory synapses observed in CA1 during fast oscillations in vitro
(Bracci et al. 1999; Whittington et al. 1997b
). Individual IPSPs were not discernible during this
protocol; they merged to create a biphasic hyperpolarizing-depolarizing waveform on which evoked EPSPs were superimposed. The EPSPs underwent a
strong dynamic modulation during repetitive stimulation, resulting in a
large polysynaptic facilitation at the end of the protocol. Changes in
synaptic inhibition, rather than in the efficacy of excitatory
synapses, were responsible for this phenomenon. Intracellular chloride
accumulation and the resulting depolarizing shift in GABAA receptor reversal potential turned out to
be the main cellular determinant of decreased efficacy of inhibitory
processes during gamma and beta frequency activation.
This study used electrical stimulation to recruit both mono- and
polysynaptic postsynaptic potentials. This was done because summation
of IPSPs and EPSPs involves a number of nonlinear processes that
complicate prediction of the effects of simultaneous activation of a
large number of synapses. During fast oscillations induced by tetanic
stimulation in the CA1 region, a large number of pyramidal neurons and
interneurons fire simultaneously on each cycle (Traub et al.
1996b; Whittington et al. 1997a
). Therefore a
large number of inhibitory and excitatory synapses are repetitively
activated on a given pyramidal neuron. Understanding the dynamic
changes induced by these events is essential to understand the
operation of the rhythmogenic network.
During the first two to four stimuli at 40 Hz, evoked EPSPs increased
in amplitude. They then strongly decreased, concomitantly with a slow
depolarization and with a large decrease in input resistance. While the
early increase in EPSP amplitude can be attributed to a genuine
potentiation of excitatory synapses (Davies and Collingridge
1996), the late decrease in amplitude is attributable to
decreased input resistance. This decrease in amplitude was accompanied
by a moderate increase in EPSP half-width, which was likely due to the
absence of a curtailing IPSP because a similar increase has been
observed for monosynaptic EPSPs as a result of paired-pulse depression
of GABAAergic potentials (Davies and Collingridge 1996
). When stimulation frequency was switched to 10 Hz, a progressive, large increase in EPSPs amplitude and duration was observed. This is attributable to recovery of pyramidal neuron input resistance and persistent decreased efficacy of synaptic inhibition, also manifest from the absence of hyperpolarizing IPSPs.
Under these conditions, polysynaptic EPSPs were strongly facilitated as
shown by their irregular, multi-peak shape and by the more than
threefold increase in their half-width. In support of this view,
GABAA antagonists abolished the EPSP
increase-decrease-increase sequence. These agents also unmasked a
moderate use-dependent depression of EPSPs at the end of the 40/10-Hz
protocol, showing that excitatory synapses did not undergo a direct
potentiation under these conditions. The observation that
GABAA antagonists also converted the slow
biphasic waveform into a paroxysmal burst during 40-Hz stimulation,
followed by a rapid repolarization during 10-Hz stimulation, led to the
conclusions that the slow waveform seen under control conditions was
due to depolarizing GABA effects and that it was causally related to
the associated modulation of the EPSPs. The present experimental
protocol did not cause long-term changes in synaptic efficacy either in
control solution or in the other pharmacological conditions, presumably
due to the features of the trains applied and the position of the
stimulating electrode.
Although depolarizing GABA effects have been observed under several
experimental conditions in the hippocampus (Andersen et al.
1980a; Avoli and Perreault 1987
;
Cherubini et al. 1991
; Grover et al.
1993
; Kaila et al. 1997
; Lambert et al.
1991
; Lamsa and Kaila 1997
; Manuel and
Davies 1998
; Smirnov et al. 1999
; Staley and Proctor 1999
; Staley et al. 1995
), the
cellular mechanisms underlying this phenomenon remain controversial.
Staley and Proctor (1999)
pointed out intracellular
chloride accumulation as the main factor for depolarizations induced by
exogenous GABA application or tetanic stimulation (200 Hz for 50 ms),
while Kaila et al. (1997)
identified extracellular
potassium accumulation as the sustaining mechanism of high-frequency
(100 Hz for 400 ms) stimulation induced depolarizations.
Our experiments addressed this issue in a different experimental
condition in which depolarizing GABA effects arise from repetitive activation of inhibitory synapses at 40 Hz. The experiments in the
presence of ionotropic glutamate and GABAB
receptor blockers allowed a pharmacological isolation of
GABAA-mediated effects. Under these conditions,
GABA release was reduced by suppression of polysynaptic IPSPs
(Davies et al. 1990), but it was favored by removal of
GABAB receptor mediated presynaptic inhibition. By adjusting the stimulation intensity, it was possible to elicit a
slow depolarization of similar amplitude to that in control solution
and to study the mechanisms underlying this event. If the slow
depolarization was sustained by extracellular potassium accumulation,
its time course would not be affected by manipulations of the recorded
neuron membrane potential. Under the present conditions, however, the
kinetics of the slow depolarization depended on the state of
polarization of the membrane. Recordings from current-clamped glial
cells, used to monitor potassium-mediated depolarization (Kaila
et al. 1997
), revealed that the 40/10-Hz protocol produced a
measurable potassium accumulation, but the time course of this phenomenon was much slower than the depolarization observed in pyramidal neurons. Thus even though extracellular potassium
accumulation directly depolarizes neurons and can affect
ECl by altering intracellular chloride
(Thompson and Gahwiler 1989
), it is unlikely to be the main factor responsible for the slow depolarization observed in the
present experiments. Similarly, the increase in polysynaptic EPSPs does
not seem to be mainly due to potassium mediated facilitation of
transmitter release because it lasted much longer than the extracellular potassium accumulation.
Numerical simulations, which included intracellular chloride changes,
accounted for the voltage dependence of the slow depolarization and of
the GABAA receptor driving force
(Em EGABA) observed during the 40/10-Hz
protocol. Experimentally, this driving force was monitored through
responses to 10-Hz pulses. These experiments revealed that when the
cell was between
60 and
40 mV, GABAA driving
force (positive at rest) became very small (i.e., did not gave rise to
detectable postsynaptic potentials) during the whole 10-Hz train and
remained so for several seconds after it; conversely, when the cell was
hyperpolarized to
95 mV, the (negative) GABAA
driving force recovered much earlier, so that already after 500 ms of
stimuli at 10-Hz GABAergic potentials were clearly detectable. In the
simulations, this behavior could be explained by considering the mixed
permeability of GABAA receptor to chloride and
bicarbonate (Staley et al. 1995
) and assuming stability
of transmembrane bicarbonate gradients (Staley and Proctor
1999
; Staley et al. 1995
). One of the key
features of the simulations was that the initial driving force for
chloride (Em
ECl) was much larger in the more
depolarized conditions and remained so during the activation of the
GABA conductance, thus allowing a more prolonged chloride accumulation
and delaying both the peak of the slow depolarization and the return of
EGABA to control values. We conclude
that, under the present conditions, intracellular chloride accumulation
(rather than extracellular potassium accumulation) was the main
determinant of the GABAergic depolarization and the associated EPSP modulation.
Whether depolarizing GABA effects are functionally excitatory or
inhibitory under different conditions is a matter for debate. Depolarizing GABA effects induced by tetanic stimulation of CA1 in the
presence of the GABA uptake inhibitor tiagabine are accompanied by a
functional reduction of evoked EPSPs (Jackson et al.
1999). Under the conditions of the present study, depolarizing
GABA effects caused a clear disinhibition of the CA1 network, favoring
polysynaptic EPSPs and converting subthreshold evoked PSPs into
superthreshold ones. As in the case of depolarizing GABA potentials
induced by tetanic stimulation (Bracci et al. 1999
),
presynaptic GABAB receptors exert an important
negative feedback by limiting the amount of GABA released and therefore
the extent of the GABAAergic depolarization.
The present results set some constraints on the models of fast
oscillations in CA1, which often rely on synchronous, rhythmic IPSPs to
explain rhythmogenesis (Fisahn et al. 1998;
Jefferys et al. 1996
; Traub et al. 1996a
;
Whittington et al. 1995
). When a large number of
inhibitory synapses impinging on pyramidal neurons are activated
simultaneously at gamma frequency, a rapid collapse of chloride
gradients takes place, resulting in a loss of inhibitory efficacy.
Stimuli were applied to the pyramidal layer, presumably activating
local interneurons and pyramidal neurons. This method does not allow,
however, a precise control over the class of interneurons excited,
either directly or synaptically. Other studies have shown that the
GABAergic synapses giving rise to depolarizing effects tend to be
located on the dendrites rather than the soma of pyramidal neurons
(Lambert et al. 1991
; Staley and Proctor
1999
). It is possible that, if only a particular subgroup of
interneurons (possibly impinging on the soma of pyramidal neurons) is
rhythmically active, less intracellular chloride accumulation might
take place, thus preserving the inhibitory efficacy of the IPSPs. This
may be the case of gamma oscillations induced by focal application of
mGluR agonists (Traub et al. 1996a
; Whittington
et al. 1995
), where a much more limited number of interneurons
and even fewer pyramidal neurons are actively involved in the
oscillation than during tetanically induced oscillations.
Intense mental activity may trigger epileptic discharges
(Matsuoka et al. 2000). Gamma oscillations have been
linked to cognitive functions (Hopfield 1995
;
Singer 1999
). These two observations lead to the
intriguing hypothesis that one possible mechanism of this
physiopathological transition might involve intracellular chloride
accumulation induced by repetitive activation of inhibitory neurons at
gamma frequency, resulting in a transient disinhibition of the local
networks and therefore in a decreased threshold for epileptic
discharges (Köhling et al. 2000
).
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ACKNOWLEDGMENTS |
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This work was supported by Wellcome Trust.
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FOOTNOTES |
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Address for reprint requests: J.G.R. Jefferys (E-mail: j.g.r.jefferys{at}bham.ac.uk).
Received 18 December 2000; accepted in final form 13 March 2001.
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REFERENCES |
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