1Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada; 2Neurological Institute, Tokyo Women's Medical College, Tokyo 162, Japan; 3Istituto di Ricovero e Cura a Carattere Scientifico Neuromed, 86077 Pozzilli (Isernia); and 4Ospedale San Paolo, Università degli Studi di Milano, 20142 Milan, Italy
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ABSTRACT |
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D'Antuono, Margherita, Hiroto Kawasaki, Carmela Palmieri, and Massimo Avoli. Network and Intrinsic Contributions to Carbachol-Induced Oscillations in the Rat Subiculum. J. Neurophysiol. 86: 1164-1178, 2001. Low-frequency network oscillations occur in several areas of the limbic system where they contribute to synaptic plasticity and mnemonic functions that are in turn modulated by cholinergic mechanisms. Here we used slices of the rat subiculum (a limbic area involved in cognitive functions) to establish how network and single neuron (intrinsic) membrane mechanisms participate to the rhythmic oscillations elicited by the cholinergic agent carbachol (CCh, 50-100 µM). We have found that CCh-induced network oscillations (intraoscillatory frequency = 5-16 Hz) are abolished by an antagonist of non-N-methyl-D-aspartate (NMDA) glutamatergic receptors (n = 6 slices) but persist during blockade of GABA receptors (n = 16). In addition, during application of glutamate and GABA receptor antagonists, single subicular cells generate burst oscillations at 2.1-6.8 Hz when depolarized with steady current injection. These intrinsic burst oscillations disappear during application of a Ca2+ channel blocker (n = 6 cells), intracellular Ca2+ chelation (n = 6), or replacement of extracellular Na+ (n = 4) but persist in recordings made with electrodes containing a blocker of voltage-gated Na+ channels (n = 7). These procedures cause similar effects on CCh-induced depolarizing plateau potentials that are contributed by a Ca2+-activated nonselective cationic conductance (ICAN). Network and intrinsic oscillations along with depolarizing plateau potentials were abolished by the muscarinic receptor antagonist atropine. In conclusion, our findings demonstrate that low-frequency oscillations in the rat subiculum rely on the muscarinic receptor-dependent activation of an intrinsic oscillatory mechanism that is presumably contributed by ICAN and are integrated within the network via non-NMDA receptor-mediated transmission.
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INTRODUCTION |
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Rhythmic, low-frequency (i.e.,
4-12 Hz) network oscillations are recorded in vivo and in vitro from
many regions of the limbic cortex (Alonso and Garcia-Austt
1987; Bland 1986
; Bland and Colom 1993
; Buzsaki and Eidelberg 1983
; Dickson
et al. 1995
; Fischer et al. 1999
;
MacVicar and Tse 1989
). These oscillations, referred to
as theta rhythm when studied in vivo, may contribute to long-lasting changes in synaptic efficacy and thus to mnemonic functions
(Buzsaki et al. 1994
; Huerta and Lisman
1993
; Larson and Lynch 1986
;
Pavlides et al. 1988
; Singer 1993
). A
common way to induce in vitro low-frequency oscillations relies on the
use of cholinergic agents (Chapman and Lacaille 1999
;
Konopacki et al. 1988
; MacVicar and Tse
1989
; McMahon et al. 1998
; Osehobo and
Andrew 1993
). Interestingly, one type of theta recorded in vivo
is sensitive to treatments that antagonize muscarinic transmission
(Kramis et al. 1975
). Cholinergic mechanisms are indeed
involved in learning and memory (Ashe and Weimberger
1991
; Dunnett and Fibiger 1993
; Winkler
et al. 1995
) as well as in the phatophysiogenesis of epileptic
discharges (Nagao et al. 1996
; Turski et al.
1989
).
It is believed that low-frequency network oscillations result from
specific oscillatory properties of the neuron membrane that are
implemented within the network through the interaction of excitatory
and inhibitory synaptic mechanisms (Bland and Colom 1993; Chapman and Lacaille 1999
; Klink
and Alonso 1997a
; McMahon et al. 1998
). It is,
however, unclear how the membrane oscillations are processed within the
network since they are often subthreshold for action potential
discharge. Moreover, the ionic mechanisms responsible for the intrinsic
oscillatory behavior remain poorly defined. For instance,
Na+ electrogenesis contributes to subthreshold
oscillations (Klink and Alonso 1993
; Mattia et
al. 1997
; Pape and Driesang 1998
), but
activation of muscarinic receptors leads to a decrease of Na+ currents (Cantrell et al.
1996
; Mittmann and Alzheimer 1998
).
In this study, we have used slices of the rat subiculum to establish
how network and single neuron (intrinsic) membrane properties contribute to the low-frequency, rhythmic oscillations elicited by the
nonhydrolizable cholinergic agent carbachol (CCh). The subiculum
represents a gating point for most of the information entering and
leaving the hippocampus proper (Amaral and Witter 1989;
Lopes da Silva et al. 1990
), and it may be involved in
cognitive functions such as spatial learning (Barnes et al.
1990
; Sharp and Green 1994
) as well as in the
spread of seizure activity within the limbic system (Lothman et
al. 1991
). Muscarinic receptor activation in the subiculum
leads to the appearance of depolarizing plateau potentials that are
caused by a Ca2+-activated nonselective cation
conductance (ICAN) (Kawasaki et al. 1999
; cf. Caesar et al. 1993
; Fraser
and MacVicar 1996
; Klink and Alonso 1997a
,b
;
Schwindt et al. 1988
). Here we report that subicular
neurons can generate synchronous oscillations at 5-16 Hz in the
presence of CCh and that these network oscillations depend on the
function of non-N-methyl-D-aspartate (NMDA)
glutamatergic receptors, while they persist with blockade of GABA
receptor-mediated transmission. In addition, we have found that during
blockade of glutamatergic (and GABAergic) transmission, single
subicular cells can produce voltage-dependent rhythmic burst
oscillations at 2.1-6.8 Hz that may be contributed by an
ICAN-like mechanism. Finally, both
network and intrinsic oscillations are abolished by the muscarinic
receptor antagonist atropine.
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METHODS |
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Brain slices were obtained from male Sprague-Dawley rats
(200-300 g), following standard procedures (Kawasaki et al.
1999; Nagao et al. 1996
). Animals were
anesthetized with halothane and decapitated. After removing the brain,
transverse slices (400-450 µm) were cut with a vibroslice apparatus.
Slices containing the whole subicular complex were transferred to a
tissue chamber and kept at an interface between oxygenated artificial
cerebrospinal fluid (ACSF) and humidified gassed (95%
O2-5% CO2) atmosphere (33 ± 1°C). ACSF composition was (in mM) 124 NaCl, 2 KCl, 1.25 KH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose (pH 7.4). The following
drugs were bath applied: atropine (0.5-1 µM), bicuculline methiodide
(BMI, 10 µM), CCh (50-100 µM, most often 70 µM),
6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 10 µM),
3-((±)-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP, 20 µM), P3-amino-propyl, P-diethoxymethylphosphonic acid
(CGP-35348, 1 mM), CoCl2 (2 mM),
(D-Ala2-N-Me-Phe, Gly-ol)enkephalin (DAGO, 10 µM),
(+)-alpha-methyl-4-carboxy-phenylglycine (MCPG, 1 mM), picrotoxin
(50-100 µM), tetrodotoxin (TTX, 1 µM), and
D-tubocurarine (D-TC, 1 µM). When
CoCl2 was used
KH2PO4 was omitted,
MgSO4 was replaced with
MgCl2, and KCl was increased from 2 to 3.25 mM. In some experiments, NaCl in the ACSF was replaced by equimolar choline. Chemicals were acquired from Tocris Cookson (Langford, UK) or
Sigma (St. Louis, MO), while CGP-35348 was a kind gift of Novartis (Basel).
Field potential recordings were made in the subiculum with ACSF-filled
glass pipettes (tip resistance = 2-6 M) that were connected
with a DC amplifier. Sharp-electrode intracellular recordings were
performed with pipettes filled with one of the following solutions: 3 or 4 M K-acetate (tip resistance = 70-120 M
); 3 M K-acetate + 0.2 M of the Ca2+ chelator
bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA; tip resistance = 70-120 M
); or 3 M K-acetate + 50 mM 2-(tri-methyl-amino)-N-(2-6-dimethyl-phenyl)-acetamide
(QX-314, a kind gift of Astra, Toronto, ON). Intracellular signals were fed to a high-impedance amplifier with internal bridge circuit for
intracellular current injection. The bridge was monitored throughout
the experiment and adjusted as required. Whenever necessary the resting
membrane potential (RMP) was kept at a given value by injecting steady
intracellular current. In some experiments, surgical cuts were made
with a micromanipulator-mounted knife to isolate the subiculum, while a
bipolar, stainless steel electrode was used to deliver extracellular
stimuli (90 µs,
2 µA) at a site that was close (300-800 µm) to
the recorded neuron. By doing so, we established whether the
stimulus-induced synaptic responses contained any excitatory component,
and thus we assessed the efficacy of the block exerted on ionotropic
excitatory transmission by CNQX and CPP (see Fig. 7B).
Extracellular and intracellular signals were displayed on an oscilloscope as well as on a Gould pen chart recorder or a Gould WindoGraf recorder. They were also stored on a video cassette recorder and/or fed to a computer interface (Digidata 1200B, Axon Instruments) for subsequent analysis made with the software pClamp8 (Axon Instruments). Average power spectra of the field potential and/or intracellular signals (duration = 7-10 s) were calculated by using the Hamming's equation with the software Clampfit8 (Axon Instruments). Power spectra of the intracellular activity generated by subicular cells were obtained after filtering the signals with a low-pass filter set to 100 Hz.
Subicular cells included in this study responded to brief (10-120 ms)
intracellular pulses of depolarizing current (<0.7 nA) by generating
bursts of action potentials. Hence they could be identified as
bursting-firing cells according to the criteria reported in previous
studies (Mattia et al. 1993, 1997
;
Stewart and Wong 1993
; Taube
1993
). The fundamental electrophysiological parameters of these
cells were measured as follows: RMP after cell withdrawal; input
resistance (Ri) from the maximum
voltage change in response to a hyperpolarizing current pulse (100-200 ms, <
0.5 nA); and action potential amplitude from the baseline. The
electrophysiological properties recorded in normal ACSF in a
representative group of subicular cells were RMP =
64.1 ± 9.0 mV (n = 26), Ri = 45.6 ± 9.9 M
(n = 26), and action potential amplitude = 70.4 ± 3.2 mV (n = 23).
Quantitative results throughout this paper are expressed as means ± SD, and n indicates the cell number. Results were
compared with the paired Student's test and were considered
significantly different if P < 0.05.
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RESULTS |
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Field potential and intracellular characteristics of carbachol-induced network oscillations
Spontaneous field potential oscillations at 5-16 Hz appeared
after ~20 min of continuous application of ACSF containing CCh. These
oscillations could persist throughout the experiment (5 h) and were
organized in series of waves riding a DC negative shift, 1-4 mV in
amplitude. The oscillatory sequences occurred at intervals of 2.5-18 s
(8.1 ± 3.2 Hz, n = 25), each lasting 0.8-8.3 s
(3.6 ± 1.6 s, n = 25; Figs.
1A and
2A, field). The plot in Fig.
1B shows that the duration and the interval of occurrence of
the oscillatory sequences recorded in different slices were directly
correlated. As illustrated in Fig. 1C, these field
oscillations became of larger amplitude when the recording electrode
was positioned at 0.8-1.5 mm from the pia and changed in polarity when
obtained near the pial surface, thus suggesting a current sink between these two recording sites. Moreover, they continued to occur
spontaneously in the subicular cortex after cutting the connections
with either the hippocampus proper (n = 5; Fig.
1D) or the entorhinal cortex (n = 7; not
illustrated). Hence these data demonstrated that neuron networks in the
subiculum responded per se to CCh by producing low-frequency field
potential oscillations similar to those reported in other limbic
structures maintained in vitro (Dickson and Alonso 1997
;
Konopacki et al. 1988
; MacVicar and Tse
1989
).
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Intracellular recordings made from subicular bursting neurons in the presence of CCh revealed that the field potential oscillations were associated with sequences of rhythmic depolarizations that reached amplitudes of 8-24 mV and could trigger either single action potentials or action potential bursts (Figs. 1A and 2A). The frequency of the field potential oscillations (and of the concomitant rhythmic depolarizations) within each sequence was higher at onset (10.8 ± 4.6 Hz, values obtained from 14 experiments during the initial 1 s) as compared with what seen at the end (7.3 ± 1.6 Hz, values calculated in the same slices during the last 1 s, P < 0.02). Such a change in frequency could be also appreciated by obtaining the power spectra of the field potential occurring at the beginning and at the end of each oscillatory sequence (Fig. 2B). Moreover, the intracellular oscillations occurring at the beginning of each sequence were characterized by a higher degree of correlation with the field potentials as well as by a more pronounced rhythmicity (Fig. 2C).
Changing the membrane potential with injection of hyperpolarizing or
depolarizing current in nine subicular cells recorded from six slices,
did not influence the occurrence of the sequences of rhythmic
oscillations or their pattern rhythmicity, further demonstrating that
the oscillations resulted from neuron network interactions. This
procedure, however, modified the amplitude of the rhythmic
depolarizations occurring during each oscillatory sequence. As
illustrated in Fig.
3A
(middle), <50% of the rhythmic depolarizations triggered
action potentials when the membrane was slightly hyperpolarized from an
RMP = 66 mV. Further hyperpolarization abolished all action
potential firing while increasing the amplitude of the rhythmic
depolarizations (Fig. 3A, bottom). Progressive hyperpolarization of the membrane also revealed a steady depolarizing envelope that occurred throughout the sequence of field potential oscillations. Conversely, depolarizing the neuron membrane with steady
injection of positive current caused sustained firing of action
potentials during the rhythmic oscillations; this action potential
discharge appeared to obscure the periods of firing silence occurring
between the rhythmic depolarizations (Fig. 3A, top).
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The relation between field potential and intracellular activity at the
different membrane potentials is further shown in the samples
illustrated in Fig. 3B where traces were superimposed by
using the negative peaks of the field potential oscillations. By
bringing the membrane potential to depolarized values, we could also
record rhythmic intracellular oscillations at 3-6 Hz that were often
associated with action potential bursts and were not mirrored by any
field potential activity (Fig. 3A, ).
Pharmacological features of the of CCh-induced network oscillations
Both field potential and intracellular oscillations induced by CCh
were unaffected by bath application of the NMDA receptor antagonist CPP
(10 µM, n = 5; not shown) but disappeared during application of the non-NMDA receptor antagonist CNQX (10 µM,
n = 6; Fig.
4A). Under
these conditions, intracellular rhythmic depolarizations continued to
occur when the membrane potential was set at slightly depolarized
levels (Fig. 4A, bottom, ). These intrinsic
oscillations, which were capable of triggering single action potentials
or action potential bursts, had a lower frequency (i.e., 3.5-7 Hz)
than those associated with field potential activity before CNQX
application (i.e., 8-16 Hz). As shown in Fig. 4A
(top,
), "asynchronous," burst oscillations were also
seen under control conditions when the membrane was depolarized with
injection of intracellular current. The power spectra illustrated in
Fig. 4B were obtained from the intracellular activity
corresponding to the asynchronous oscillations (a) and to
the network oscillations (b) recorded under control
conditions as well as to the asynchronous oscillations seen during CNQX
(c).
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CCh-induced rhythmic oscillations continued to occur during
concomitant application of the GABAA and
GABAB receptor antagonists BMI (10 µM) and CGP
35348 (1 mM; n = 11; Fig.
5). The oscillations recorded in the
presence of BMI and CGP 35348 were of longer duration and had longer
intervals of occurrence (P < 0.02; Fig. 5,
B and C). Moreover, the longer duration was
characterized by a larger amount of low oscillation frequency as
compared with what seen under control conditions (P < 0.05; Fig. 5A). Since BMI may influence Ca2+-dependent K+
conductance (Debarbieux et al. 1998), we also analyzed
in five slices the effects induced on the network oscillations by
application of the GABAA receptor antagonist
picrotoxin (50-100 µM) along with the GABAB
receptor antagonist CGP 35348 (1 mM). As shown in Fig.
6A, CCh-induced oscillations
continued to occur in the presence of picrotoxin. However, in contrast
to what observed with BMI, picrotoxin decreased the duration
(P < 0.02) and increased the interval of occurrence
(P < 0.05) of the oscillatory sequences (Fig.
6C, a and b). Moreover, picrotoxin did not change
significantly the frequency of the oscillations occurring within each
sequence (Fig. 6, B and Ca). Activation of
µ-opioid receptors by DAGO (10 µM; n = 4), a
procedure that presumably blocks GABA release (Capogna et
al. 1993
) did not cause any consistent change in the
oscillatory pattern (not illustrated).
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The field potential rhythmic oscillations induced by CCh were abolished by application of the muscarinic receptor antagonist atropine (0.5 µM, n = 5; not illustrated) but were not influenced by the nicotinic receptor antagonist D-TC (1 µM, n = 4; not illustrated) or by the metabotropic glutamate receptor antagonist MCPG (1 mM; n = 3; not illustrated).
Voltage-dependent burst oscillations induced by CCh during blockade of excitatory transmission
The ability of subicular cells to generate "asynchronous" membrane oscillations in the presence of CCh during steady membrane depolarization and the occurrence of a similar oscillatory pattern during application of CNQX suggested to us that intrinsic membrane mechanisms might contribute to the oscillations expressed by the subicular network under control conditions. Hence we studied the ability of subicular bursting cells to generate oscillations during application of CCh and excitatory amino acid receptor antagonists. We assumed that under these conditions, ionotropic excitatory synaptic transmission was not operant since only isolated inhibitory postsynaptic potentials (IPSPs) were induced by extracellular focal stimuli (Fig. 7B).
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First, we analyzed the effects induced by CCh on the intrinsic
oscillations generated by 26 subicular neurons during blockade of
excitatory transmission (cf. Mattia et al. 1997). As
illustrated in Fig. 7A1 (left), steady
depolarizing commands positive to
65 mV made subicular neurons
recorded during application of CNQX and CPP, generate persistent
subthreshold membrane oscillations at 4.8-6.3 Hz (Mattia et al.
1997
). These oscillations were voltage dependent since further
depolarization to approximately
60 mV caused an increase in their
amplitude, and the occasional occurrence of single action potentials
(Fig. 7A1, *). Subicular neurons also generated clusters of
single action potentials when the membrane potential was brought to
values more positive than the level dominated by the subthreshold
oscillations (not illustrated) (but see Mattia et al.
1997
).
CCh addition caused a small, marginally significant (5.4 ± 3.3 mV, n = 24), steady depolarization of the RMP, a
nonsignificant increase of the membrane input resistance measured at
membrane values equal to control RMPs (from 45.6 ± 9.9 to
48.5 ± 12.0 M, n = 26) and a decrease of the
afterhyperpolarizations (not shown) (but cf. Kawasaki et al.
1999
). Moreover during CCh application, steady depolarization
to membrane values similar to those used under control conditions,
elicited subthreshold membrane oscillations that were at times lower in
frequency and significantly smaller in amplitude than in control (Fig.
7, A1 and C). However, under these conditions,
further membrane depolarization caused the appearance of a series of
action potential bursts that repeated at 3.1 ± 0.7 Hz
(n = 31; range = 2.1-6.8 Hz) and were separated by
hyperpolarizations with amplitudes
18 mV and durations
ranging 100-280 ms (Fig. 7A1, right, and A2).
The spontaneous recurrent bursts recorded in ACSF containing CCh + CCP + CNQX could be initiated by a discharge of action potentials
(Fig. 7A2), by a series of membrane oscillations (Fig. 8A), or by a depolarizing
plateau potential triggered by a brief depolarizing pulse of
intracellular current (not illustrated). Recurrent burst oscillations
could repeat for several seconds until the action potential firing
became tonic. Such transition in firing modality was accompanied by
disappearance of the interburst hyperpolarizations. Isolated
hyperpolarizing potentials (amplitude = 4-8 mV, duration = 80-170 ms) that were not immediately preceded by any action potential,
and thus represented presumptive spontaneous IPSPs, were also seen at
RMP or during the spontaneous recent bursts (Fig. 8A, ).
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Pharmacological features of the voltage-dependent burst oscillations induced by CCh
The ability of subicular cells to generate recurrent burst oscillations in the presence of CNQX and CPP indicates that this type of CCh-induced neuron activity is not contributed by the activation of ionotropic excitatory amino acid receptors. We also established whether inhibitory conductances participated to these intrinsic, recurrent burst oscillations by bath applying the GABAA and GABAB receptor antagonists BMI (10 µM) and CGP-35348 (1 mM) to medium containing CCh + CCP + CNQX (n = 4). As shown in Fig. 8B, blockade of these GABA receptors did not influence the CCh-induced bursting oscillations but abolished both stimulus-induced and spontaneous hyperpolarizing potentials (not illustrated).
Recurrent burst oscillations induced by application of medium containing either CCh + CCP + CNQX or CCh + CPP + CNQX + BMI + CGP 35348 were abolished by the muscarinic receptor antagonist atropine (1 µM, n = 7; Fig. 8C). By contrast, they were not affected by treatment with either D-TC (1 µM, n = 4) or MCPG (1 mM, n = 3; not illustrated).
Ca2+ dependence of the voltage-dependent burst oscillations induced by CCh
CCh-induced recurrent bursting was not seen during
application of the Ca2+ channel antagonists
Co2+ (2 mM, n = 6), which
abolished stimulus-induced synaptic responses (not illustrated). As
shown in Fig. 9A, steady
depolarization of the neuron membrane to values more positive that
those capable of inducing bursting oscillations under control
conditions (i.e., in medium containing CNQX + CPP + CCh) caused
the appearance of subthreshold oscillations that had shape and
frequency similar to those seen before (4.8-6 Hz) application of CCh.
In addition, bath application of Co2+ abolished
the ability of subicular cells treated with CCh to generate
depolarizing plateau potentials (not shown) (but see Kawasaki et
al. 1999).
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Next we used electrodes filled with K-acetate and the
Ca2+ chelator BAPTA to analyze the intrinsic,
recurrent bursting induced by application of CCh and excitatory amino
acid receptor antagonists (n = 6). During the first
(12) minutes of intracellular recording, cells recorded with
BAPTA-filled electrodes generated both bursting oscillations during
steady membrane depolarization and depolarizing plateau potentials in
response to brief depolarizing pulses (Fig. 9B, +5 min).
Later (i.e., >15 min after impalement), a progressive blockade of both
types of activity ensued. Under these conditions, subicular cells that
were steadily depolarized could oscillate at frequencies (i.e.,
5.9 ± 09 Hz, n = 5) within the range of those
seen before CCh application. In addition, brief pulses of intracellular
depolarizing current only induced action potential bursts (Fig.
9Bb). The plot in Fig. 9C summarizes the
frequency values of the oscillations recorded under different
conditions that were aimed at reducing Ca2+
activity during application of CCh.
Na+-mediated conductances and carbachol-induced intrinsic burst oscillations
The burst oscillations recorded in medium containing
CCh and excitatory amino acid receptor antagonists were abolished by addition of the Na+ channel blocker TTX (1 µM,
n = 5). As reported in previous studies performed in
the subiculum (Kawasaki et al. 1999; Mattia et
al. 1993
, 1997
), the effects induced by TTX were characterized
by blockade of all regenerative activity including plateau potentials and action potential bursts induced by brief depolarizing current pulses (not illustrated). These results suggested that the TTX-induced effects could reflect the inability of depolarizing commands to influence neuronal membrane compartments (e.g., dendrites) that were
remote to the recording site (see also Fraser and MacVicar 1996
). Hence, we used QX-314-filled electrodes to analyze the involvement of voltage-gated, Na+ electrogenesis
in CCh-induced intrinsic burst oscillations. Intracellular application
of QX-314 (or of other lidocaine quaternary derivatives) blocks
voltage-gated Na+ channels and also depresses
K+ conductances (Andreasen and Hablitz
1993
; Mulle et al. 1985
), thus allowing a better
electrotonic somatodendritic transfer.
As illustrated in Fig. 10, the recurrent burst oscillations induced by CCh changed in shape during intracellular recordings performed with QX-314-filled electrodes (n = 7). In particular, fast action potential burst riding over the oscillations were replaced over time by slow events representing presumptive Ca2+ spikes. In spite of these changes, subicular cells recorded with QX-314-filled electrodes continued to generate slow oscillatory events in response to steady depolarizing commands (Fig. 10Aa). In addition the frequency of the CCh-induced oscillatory pattern was not significantly changed (Fig. 10Aa). Finally, under these recording conditions brief pulses of depolarizing current continued to induce depolarizing plateau potentials (Fig. 10Ab).
|
In four of the seven subicular cells recorded with QX-314-filled electrodes, we analyzed the effects induced by extracellular Na+ replacement on CCh-induced oscillations and depolarizing plateau potentials. As shown in Fig. 10A, application of choline-containing medium abolished the slow membrane oscillations elicited by steady membrane depolarizations (a) along with the depolarizing pulse-triggered plateau potentials (b). A quantitative summary of the results obtained with QX-314-filled electrodes before, during, and after Na+ replacement is illustrated in Fig. 10B. It is evident from this plot that CCh-induced oscillations and depolarizing plateau potentials in subicular neurons were changed in a similar fashion by extracellular Na+ replacement.
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DISCUSSION |
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We have found that bath application of CCh to slices of the rat subiculum induces network-driven oscillations that occur at 5-16 Hz and are caused by the activation of muscarinic receptors. This type of activity depends on the function of non-NMDA glutamatergic receptor synchronizing mechanisms and is enhanced rather than abolished, by blockade of GABAA and GABAB receptors. In addition, we have discovered that during CCh treatment subicular cells generate voltage-dependent rhythmic burst oscillations that repeat at 2.1-6.8 Hz even when ionotropic excitatory transmission is abolished. This CCh-induced burst activity also depends on muscarinic receptor activation and is presumably contributed by a nonselective cationic conductance that is instrumental in the generation of depolarizing plateau potentials as well.
CCh-induced, network-driven oscillations in the subiculum
Rhythmic oscillations of neuron populations can be recorded in
vitro from limbic structures (Dickson and Alonso 1997;
Fischer et al. 1999
; Konopacki et al.
1988
; MacVicar and Tse 1989
; Osehobo and
Andrew 1993
) as well as from the neocortex (Lukatch and
Maciver 1997
) during application of cholinergic agents. The
field potential characteristics of the oscillations recorded in the rat
subiculum share many similarities with those reported in these previous studies, including intraoscillation frequency, organization of the
pattern in sequences lasting
8 s and sensitivity to the muscarinic receptor antagonist atropine. In addition, these oscillations continue
to occur during application of nicotinic or metabotropic glutamate
receptor antagonists (Taylor et al. 1995
).
The oscillations induced in the subiculum by CCh are synaptically
driven and rely on glutamatergic transmission mediated through non-NMDA
receptors. This conclusion rests on electrophysiological (i.e.,
amplitude changes during injection of hyperpolarizing or depolarizing
current) and pharmacological evidence (i.e., effects of selective
receptor antagonists), and it is in line with experimental findings
obtained in both hippocampus (MacVicar and Tse 1989) and
entorhinal cortex (Dickson and Alonso 1997
) as well as
with computer-simulation data (Traub et al. 1992
).
Moreover, the glutamatergic network underlying these rhythmic
oscillations resides in the subiculum as suggested by their persistence
following surgical isolation of this structure from the hippocampus
proper or the entorhinal cortex. Interestingly, the intracellular
rhythmic depolarizations induced by CCh were rarely associated with
action potential bursts, and firing was aborted by small membrane
hyperpolarizations. Hence, as proposed in the modeling study of
Traub et al. (1992)
, subicular CCh-triggered network
oscillations are presumably generated within a limited range of
increased excitatory synaptic conductance.
A further similarity of our findings with some published studies
(MacVicar and Tse 1989; Traub et al.
1992
) resides in the ability of network oscillations to occur
during blockade of GABA receptor-mediated inhibition or activation of
µ-opioid receptor, which leads to inhibition of GABA release from
interneuron terminals (Capogna et al. 1993
). It should
be also mentioned that CCh-induced network oscillations occur in
neocortical slices only after bicuculline application (Lukatch
and Maciver 1997
) and that intra-hippocampal injection of CCh + bicuculline elicits theta-like oscillations in vivo (Colom et
al. 1991
). However, these previous data, as well as our own
findings, are at odds with studies indicating that
GABAA receptor function may be necessary for
maintaining the repetitive structure of CCh-induced oscillations
(White et al. 2000
; Williams and Kauer
1997
). In addition, different classes of hippocampal
interneurons have been shown to pace CCh-induced rhythmic activity in
pyramidal cells (Chapman and Lacaille 1999
; McMahon et al. 1998
). It is unlikely that our
pharmacological procedures failed to block GABAA
and GABAB receptors in the subiculum network.
Rather our findings, along with evidence obtained in experiments in
which theta-like oscillations were recorded in the presence of
GABAA receptor antagonists (Colom et al.
1991
; Lukatch and Maciver 1997
; MacVicar
and Tse 1989
), suggest that IPSPs resulting from interneuron
activity are not essential for the occurrence of CCh-induced
network-driven oscillations.
Mechanisms underlying CCh-induced intrinsic oscillatory bursts
CCh application during blockade of ionotropic
glutamatergic transmission reduces the subthreshold oscillations
generated by subicular neurons (cf. Klink and Alonso
1997a,b
), presumably through a reduction of voltage-gated
Na+ currents (Cantrell et al.
1996
). Indeed, Mittmann and Alzheimer (1998)
have demonstrated that muscarinic activation inhibits a persistent
Na+ current in rat neocortical pyramidal cells.
However, in our experiments, this effect is accompanied by the
appearance of voltage-dependent burst oscillations that recur at
2.1-6.8 Hz and are dependent on the activation of muscarinic
receptors. Similar asynchronous oscillatory activity could also occur
during steady membrane potential depolarization in slices that
generated CCh-induced network oscillations.
It has been proposed that action potential bursts play a role in
synaptic plasticity and information processing (Lisman
1997). In our case the voltage-dependent intrinsic burst rhythm
may represent a strong factor in establishing network oscillatory
activity in the subiculum when synaptic excitation is operant. Moreover
the different frequency that characterizes intrinsic and network
oscillations suggests that excitatory (and inhibitory) interactions
within subicular networks shape the oscillatory pattern at a higher
rate. In keeping with this view, the initial portion of the network oscillations was characterized by a higher frequency and by a higher
degree of synchronization between intracellular and field potential signals.
Muscarinic receptor-dependent, intrinsic burst oscillations were
abolished by the voltage-gated Ca2+ channels
blocker Co2+, indicating that they depend on
Ca2+ entry. In addition, they progressively
disappeared when recordings were made with intracellular electrodes
containing the Ca2+ chelator BAPTA. This evidence
indicates that the intrinsic burst oscillations induced by CCh in the
subiculum may be dependent on voltage-gated Ca2+
entry as well as on Ca2+ release from
intracellular stores. This conclusion is in line with findings showing
that muscarinic activation leads to Ca2+
mobilization from intracellular stores via an intracellular messenger cascade (Berridge and Irvine 1989; McKinney
1993
).
We have also found that the intrinsic burst oscillations induced by CCh
are abolished by the voltage-gated Na+ channel
blocker TTX. However, this effect is unlikely to reflect the dependence
of CCh-induced oscillations on voltage-gated Na+
conductances. Rather it rests on the inability of subicular bursting cells, which are endowed of pronounced voltage-gated
Na+ electrogenesis (Mattia et al. 1993,
1997
), to produce in the presence of TTX regenerative events
capable of depolarizing sites (e.g., the dendrites) remote to the soma.
In line with this view, subicular cells recorded with QX-314-filled
electrodes generated intrinsic voltage-gated oscillations associated
with slow, presumptive Ca2+ spikes, while fast
action potentials were blocked. QX-314 abolishes voltage-gated
Na+ channel activity but also reduces
K+ currents and increases the input resistance of
the recorded neuron (Andreasen and Hablitz 1993
;
Mulle et al. 1985
), thus allowing depolarizing commands
delivered within the soma to influence dendritic compartments. Hence,
while action potential bursts depend on voltage-gated Na+ electrogenesis, our data indicate that these
conductances are not required for the generation of slow rhythmic
oscillations recorded in subicular neurons during muscarinic receptor activation.
By contrast, intrinsic, slow oscillatory activity induced by CCh was
not triggered by depolarizing subicular cells during replacement of
extracellular Na+ with choline. In these
experiments as well, we used QX-314-filled electrodes to depolarize the
neuron membrane in regions that were presumably remote to the recording
site. Moreover, during extracellular Na+
replacement, oscillations were not triggered even by injecting very
large depolarizing currents. Therefore a TTX-insensitive influx of
Na+ along with other cations such as
Ca2+ is necessary for the occurrence of the
muscarinic receptor-dependent, intrinsic, slow oscillations. Overall
this evidence suggests that the intrinsic burst oscillations generated
by subicular neurons in medium containing CCh and glutamatergic
antagonists may be due to a Ca2+ activated,
nonselective cationic conductance (also termed
ICAN). This mechanism is responsible
for the occurrence of depolarizing afterpotentials and depolarizing
plateau potentials in subicular (Kawasaki et al. 1999),
hippocampal (Caeser et al. 1993
; Fraser and
MacVicar 1996
), entorhinal (Klink and Alonso
1997a
,b
), and neocortical (Haj-Dahmane and Andrade
1996
; Schwindt et al. 1988
) neurons. In line
with this view, we have found that block of the CCh-induced intrinsic
burst oscillations by Co2+, choline or during
recordings with BAPTA-filled electrodes is paralleled by a similar
effect on the depolarizing plateau potentials generated in response to
brief pulses of depolarizing current.
Functional relevance of the low frequency oscillatory rhythms induced by CCh
It is debated whether the oscillatory activity induced by
pharmacological (mostly cholinergic) manipulations in brain slices maintained in vitro represents an in vitro model of the
electroencephalographic theta rhythm recorded in limbic areas during
specific behavioral states (Traub et al. 1992).
Regardless of the possible similarities that may exist between in vitro
and in vivo low-frequency oscillations, there is general agreement on
the contribution played by neuron membrane oscillations in
synchronizing neuron populations, in implementing functional
co-operativity and thus in ensuring synaptic plasticity (Buzsaki
et al. 1994
; Huerta and Lisman 1993
;
Larson and Lynch 1986
; Pavlides et al.
1988
). Hence the cellular and membrane mechanisms identified in
this study may contribute to muscarinic receptor-dependent oscillatory
behavior involved in synaptic plasticity. In addition, these activity
patterns may be also related to epileptogenesis, in particular to the
occurrence of seizures in patients suffering of temporal lobe epilepsy.
Such a conclusion has been put forward in the entorhinal cortex
(Dickson and Alonso 1997
) and may also apply to the
subiculum given its strategic position within the limbic system.
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ACKNOWLEDGMENTS |
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We thank Drs. D. Fraser, D. Paré, and B. McVicar for critical comments and helpful discussion and T. Papadopoulos for editorial assistance.
This work was supported by grants from the Canadian Institutes of Health Research (MT-8109), the Savoy Foundation, and the Fragile X Research Foundation of Canada.
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FOOTNOTES |
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Address for reprint requests: M. Avoli, 3801 University St., Montreal, Quebec H3A 2B4, Canada (E-mail: mavoli{at}po-box.mcgill.ca).
Received 28 September 2000; accepted in final form 16 May 2001.
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