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Vol. 59, Issue 4, 814-824, April 2001
-Aminobutyric AcidA Receptors in Hippocampal Neurons
Departments of Physiology (D.B., G.Z., M.F.J., J.F.M., B.A.O.) and Pharmaceutical Sciences (P.P.), University of Toronto, Toronto, Ontario, Canada; and Department of Anesthesia, Sunnybrook and Women's Health Science Centre, Toronto, Ontario, Canada (B.A.O.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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-Aminobutyric acid (GABA), the principal inhibitory
neurotransmitter, activates a persistent low amplitude tonic current in
several brain regions in addition to conventional synaptic currents.
Here we demonstrate that GABAA receptors mediating the tonic current in hippocampal neurons exhibit functional and
pharmacological properties different from those of quantal synaptic
currents. Patch-clamp techniques were used to characterize miniature
inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic
current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. The competitive
GABAA receptor antagonists, bicuculline and picrotoxin,
blocked both the mIPSCs and the tonic current. In contrast, mIPSCs but
not the tonic current were inhibited by gabazine (SR-95531).
Coapplication experiments and computer simulations revealed that
gabazine bound to the receptors responsible for the tonic current but
did not prevent channel activation. However, gabazine competitively
inhibited bicuculline blockade. The unitary conductance of the
GABAA receptors underlying the tonic current (~6 pS) was
less than the main conductance of channels activated during quantal
synaptic transmission (~15-30 pS). Furthermore, compounds that
potentiate GABAA receptor function including the
benzodiazepine, midazolam, and anesthetic, propofol, prolonged the
duration of mIPSCs and increased tonic current amplitude in cultured
neurons to different extents. Clinically-relevant concentrations of
midazolam and propofol caused a greater increase in tonic current
compared with mIPSCs, as measured by total charge transfer. In summary,
the receptors underlying the tonic current are functionally and
pharmacologically distinct from quantally activated synaptic receptors
and these receptors represent a novel target for neurodepressive drugs.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-Aminobutyric acid (GABA),
the major inhibitory neurotransmitter in the central nervous system,
modifies electrical activity in the brain by regulating membrane
hyperpolarization and the "shunting" of excitatory input. GABA
released from presynaptic terminal binds to GABAA
receptors clustered at the postsynaptic membrane and activates
inhibitory postsynaptic currents (IPSCs). In addition to conventional
quantal synaptic transmission, a persistent form of GABAergic
inhibition has been described in several brain regions. A small but
significant tonic GABAergic current has been observed in the cerebellum
(Brickley et al., 1996
; Wall and Usowicz 1997), cortex (Salin and
Prince, 1996), thalamus (Liu et al., 1995), and hippocampus (Otis et
al., 1991). This tonic current has been best characterized in the
cerebellum, where glomerular structures that surround synapses onto
granule cells serve as a repository for transmitter released from
neighboring synapses. Transmitter in the glomerulus may activate
high-affinity GABAA receptors with minimal
desensitization properties that are located in perisomatic and
extrasynaptic regions of granule cells (Rossi and Hamann, 1998).
The mechanisms that regulate the tonic GABAergic inhibition in other
brain regions are not well understood. The tonic conductance in the
hippocampus may result from the summation of overlapping miniature
IPSCs (Soltesz et al., 1995; Salin and Prince, 1996), or the spill-over
of vesicular transmitter released from neighboring synapses (Brickley
et al., 1996; Rossi and Hamann, 1998). Recently, it was postulated that
the tonic current results from the release of GABA from a surface
matrix reservoir that becomes exposed during exocytosis (Vautrin et
al., 2000). Also, reverse operation of GABA cotransporters (Gaspary et
al., 1998) or release of GABA from astrocytes (Liu et al., 2000) might
elevate GABA to concentrations sufficient to activate receptors. The in
vivo ambient concentration of GABA in the extracellular space, measured
using microdialysis (0.8-2.9 µM), is sufficient to activate
GABAA receptors (Lerma et al., 1986).
Alternatively, the tonic current might result from spontaneous openings
of constitutively active GABAA channels (Neelands et al., 1999; Birnir et al., 2000).
Regardless of the source of GABA responsible for the tonic current,
receptors that mediate this persistent GABAergic conductance are of
considerable physiological and pharmacological interest. Small but
persistent increases in chloride conductance alter input resistance and
membrane time constants; these changes, in turn, modulate synaptic
efficacy and synaptic integration. The tonic GABAergic current may also
play an important role in the manifestation of disease processes.
Certain types of seizures are associated with a decrease in ambient
concentrations of GABA and seizure control improves with treatments
that increase the concentration of GABA. Modulation of tonic receptors
represents a promising strategy for the development of new
anticonvulsant, anxiolytic, and anesthetic drugs. Notably, allosteric
modulation of GABAA receptor function by many
compounds strongly depends on the occupancy of the receptor by GABA, as
well as the state of receptor activation. The greatest increase in
GABAA receptor activity by benzodiazepines and
anesthetics occurs when receptors are activated by low concentrations of GABA (Harris et al., 1995). Accordingly, it is predicted that receptors underlying the tonic current (activated by low concentrations of GABA) would respond to pharmacological agents differently from receptors activated during quantal synaptic transmission.
Given the potential physiological and therapeutic importance of
GABAA receptors that mediate the tonic GABAergic
inhibition, we investigated the tonic current in hippocampal neurons.
We demonstrate the differential pharmacological properties of tonic and
synaptic currents mediated by GABAA receptors.
Midazolam and propofol produced a greater increase in charge transfer
associated with the tonic current compared with that associated with
miniature IPSCs. At concentrations that produce equivalent prolongation
of IPSCs, the anesthetic propofol had a greater effect on the tonic
current than the sedative midazolam. We speculate that modulation of
the tonic current may account for differences in the clinical actions of these two classes of compounds. Some of the results were published in abstract form (Bai et al., 1998).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Culture and Electrophysiological Techniques.
Primary
cultures of hippocampal neurons were prepared from embryonic Swiss
White mice using aseptic techniques (MacDonald et al., 1989). Cells
were maintained in culture for 13 to 18 days before use.
60 mV. The
extracellular recording solution contained 140 mM NaCl, 1.3 mM
CaCl2, 5.4 mM KCl, 2 mM
MgCl2, 25 mM HEPES, and 33 glucose, with pH
adjusted to 7.4 with 1 M NaOH. Tetrodotoxin (TTX, 300 nM) was added to
the extracellular solution to block voltage-sensitive Na+ channels, and
6-cyano-2,3-dihydroxy-7-nitroquinoxaline (10 µM) and
2-amino-5-phosphonovalerate (40 µM) were added to inhibit ionotropic
glutamate receptors. Recording electrodes were filled with a solution
containing 120 mM CsCl, 30 mM HEPES, 11 mM EGTA, 2 mM
MgCl2, 1 mM CaCl2, and 4 mM
MgATP; pH was adjusted to 7.3 with CsOH. Currents were recorded
simultaneously on a chart recorder and videotape recorder through a
digital converter and a PC computer using Strathclyde
Electrophysiological Software (SCAN or SPAN; Strathclyde
Electrophysiological Software, courtesy of Dr. J. Dempster, Strathclyde
University, United Kingdom;
http://www.strath.ac.uk/Departments/PhysPharm/ses.htm). Control and drug-containing solutions were delivered to the cultured neurons through glass barrels that were positioned close to the soma of
the neuron. Propofol was prepared from Diprivan 1% (Zeneca Pharma,
Mississauga, Ontario, Canada) and the solutions for the control
experiments contained equivalent concentrations of Intralipid (KabiVitrum Canada Inc., Toronto, Canada). Intralipid did not influence
the mIPSCs or tonic current. Midazolam was prepared from a commercial
preparation of Versed (Hoffman-LaRoche Ltd., Mississauga, Ontario,
Canada). We observed no differences in the actions of midazolam
prepared from Versed compared with the pure compound (generously
provided by Hoffman-La Roche, Nutley, NJ) dissolved in dimethyl
sulfoxide. Bicuculline methobromide was purchased from Sigma (Oakville,
Ontario, Canada) and gabazine (also known as SR-95531) was obtained
from Research Biochemical International (RBI, Natick, MA).
Whole-cell recordings were also made from the CA1 region of hippocampal
slices obtained from 2- to 3-week old Wistar rats. Coronal slices were
prepared with a vibratome (VT1000E; Leica, Wetzlar, Germany) and
incubated at room temperature for a minimum of 1 h in oxygenated
(95% O2/5% CO2)
artificial cerebrospinal fluid containing 124 mM NaCl, 3 mM KCl, 4 mM
CaCl2, 4 mM MgCl2, 26 mM
NaHCO3, 1.25 mM
NaH2PO4, and 10 mM glucose.
Slices were then transferred to a tissue chamber as needed and
maintained at 31°C ± 0.5°C at the interface between
humidified and oxygenated (95% O2/5%
CO2) aCSF perfused through the chamber at a rate
of 0.5 to 1 ml/min. Tight-seal (>5 G
) whole-cell recordings were obtained from CA1 pyramidal cells using a "blind" approach. The internal pipette solution consisted of 140 mM CsCl, 10 mM HEPES, 2 mM
MgCl2 (pH 7.2-7.3 using CsOH; osmolarity,
270-280 mOsM). Spontaneous miniature IPSCs (see below) were isolated
by the addition of 0.5 µM TTX, 10 µM
6-cyano-2,3-dihydroxy-7-nitroquinoxaline and 40 µM
2-amino-5-phosphonovalerate to the aCSF. Drugs tested were dissolved in
aCSF and superfused over slices. Spontaneous mIPSCs were recorded using
an Axopatch-1D (Axon Instruments, Foster City, CA), filtered at 2 kHz
and stored on videotape for subsequent off-line analysis using a
digital data recorder (VR-10B; InstruTECH Corp., Port Washington, NY).
Data Analysis. Current recordings that demonstrated a stable baseline and distinct mIPSCs were used for the analysis. All experiments were digitized (2 kHz) with a pulse-code modulator and stored on VHS videotapes. For analysis, the recordings were played back and re-digitized using an event detection program (SCAN). For detection of IPSCs, the trigger level was set at approximately three times higher than the level of the baseline noise (~ 3.4 pA). All events greater than the threshold level were recorded for frequency analysis including those infrequent compound events (<2%) with multiple peaks. When multiple peaks were clearly evident during the visual inspection of the records, the additional peaks were counted as mIPSCs. However, compound events were excluded from the analysis of rise time or decay of synaptic currents. In addition, we manually scrolled through files of detected events to reject spurious events that were caused by excessive noise.
Spontaneous postsynaptic currents recorded in the presence of tetrodotoxin (TTX) are referred to as miniature IPSCs (mIPSCs). Miniature IPSCs with a rapid onset (10 to 90% rise time < 5 ms) and decay phase that were not contaminated by other mIPSCs were selected for further kinetic analysis. At least 100 individual mIPSC events were recorded under each experimental condition. Peak amplitude, charge transfer (Q, the integrated area under mIPSCs), and the time constant of current decay (
off) were analyzed. The decay phase was well described by a single exponential equation in
the form I(t) = Aoexp
(t/off) + C, where
I(t) is the current amplitude at any given time t,
C is the residual current, and Ao is the current amplitude at time 0. Change in the charge transfer (
QmIPSC)
associated with mIPSC was analyzed according to Brickley et al. (1996)
using the equation QmIPSC = fdrug × Qdrug fcon × Qcon, where
fdrug and fcon
are the frequencies (Hz) of mIPSCs and Qdrug and
Qcon are the average charge transfer (pC)
per mIPSC during drug and control conditions, respectively. Under our
experimental conditions, we assumed that the change in charge transfer
reflected a proportional change in membrane conductance. The amplitude
of the tonic current was calculated as the difference between the holding current measured before and after the application of
bicuculline (10 µM) (Brickley et al., 1996; Wall and Usowicz, 1997).
The increase in the tonic current that was observed after the
application of midazolam or propofol was measured from the chart record
(Astro-Med, West Warwick, RI). The charge transfer associated with the
tonic current was calculated according the equation:
QTC = ITC × t, where QTC is the charge
transfer produced by the tonic current, ITC is
the current amplitude at steady-state, and t is time.
Variance analysis was used to estimate the single channel current
(i) from the mean current (Imean) and
current variance (
2). Variance
(2) was calculated according to the formula:
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2 Imean follows a
parabolic relationship: 2 = i(1 Po)Imean, where
Po is the channel open probability, which varies
from 0 to 1. If we assume that the channel open probability of
receptors mediating the tonic current is small under our experimental conditions (concentrations of exogenous GABA = 0.1 1 µM), then the following equation holds: i = 2/Imean. Single
channel conductance () was estimated according to the equation:
= i/(VH VR), where VH
is the holding potential and VR is the
reversal potential for chloride.
After establishing the whole-cell configuration, 10 - 20 min were
allowed to elapse before the application of drug to allow the membrane
patch to stabilize and exchange of ions between the recording electrode
and the cytosol to occur. Under these conditions, the frequency of
mIPSCs remained stable. In six cells, the frequency of mIPSCs was
measured during the first minute of recording (0.67 ± 0.08 Hz)
and 10 min later (0.68 ± 0.09 Hz). Thus, the frequency of the
mIPSCs was stable before the application of the drugs (102 ± 10%, n = 6, P = 0.93).
Simulation. A general simulator program, Axon Engineer (Aeon Software, Fort Lauderdale, FL; http://www.pompano.net/~aeonsoft/) was used to simulate the data. This program allows kinetic states to be defined and linked together by rate constants that can be a function of voltage, ion, and drug concentration. The differential equations implicit in the kinetic scheme are then integrated and driven by user-defined stimuli. The distribution of states in time is converted to open probability by assigning conductance weights to the individual states and summing the system at each time point.
Statistics. Results are presented as mean ± S.E.M. Differences between groups are considered significant for P < 0.05, using a paired Student's t test, unless otherwise indicated.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characteristics of mIPSCs and the Tonic Current in Cultured
Hippocampal Neurons.
Minature IPSCs (Fig.
1A) recorded using whole-cell methods had
a mean amplitude of 40.8 ± 2.1 pA (n = 44 neurons) at frequencies ranging from 0.06 to 2.5 Hz (0.61 ± 0.09 Hz). The mIPSCs had a rapid onset (10 to 90%; rise time, 2.4 ± 0.1 ms; n = 44) then decayed with a time course that
was generally well fit by a single exponential function
(decay = 30.9 ± 1.1 ms). Under control
conditions, the frequency of mIPSCs remained constant over the 10 min
before drug application. In addition to the transient postsynaptic
currents, a persistent or tonic current was revealed after the
application of bicuculline (Fig. 1A). Bicuculline (10 µM)
consistently caused an outward current as indicated by an 18.1 ± 1.0 pA, (n = 40) outward shift in the holding current.
Bicuculline also reduced the variance of the baseline noise from
11.8 ± 0.9 pA2 to 6.3 ± 0.5 pA2 (n = 9; P < 0.01) suggesting that the outward current was in fact caused by the
inhibition of a tonic inward current. The tonic current was attributed
to activation of GABAA receptors (Valeyev et al.,
1993) because it was also inhibited by another
GABAA receptor antagonist, picrotoxin (100 µM;
19 ± 3 pA; n = 7), and reversed polarity close to
the Nernst potential for chloride ions (3.0 ± 7 mV;
n = 6). This 20 pA current is ~0.6% of the maximum
current recorded in these cells (Orser et al., 1994) and represents
activation of ~0.4% of the receptors (Bai et al., 1999).
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) that the tonic
GABAergic current in hippocampal neurons was reduced in amplitude when
cells were perfused with a stream of saline, suggesting that a
diffusable ligand activated the persistent chloride conductance. Under
our experimental conditions, we have observed a similar phenomenon. To
avoid fluctuations in the ambient concentration of GABA, a constant low
perfusion rate was maintained throughout the experiments.
To investigate the biophysical properties of the
GABAA receptors underlying the tonic current, the
mean elementary conductance of the channels () was estimated from
the relationship: = 2/[Imean × (VH VR)].
This elementary conductance was then compared with the value for
current activated by low concentrations of exogenous GABA (0.1-1
µM). The relationship between mean current amplitude and current
variance is illustrated in Fig. 1, B and C. The unitary conductance for
the tonic current was ~5.6 pS. This value was similar to the unitary
conductance, estimated in the same way, for GABAA
receptors activated by low concentrations of exogenous GABA (~6.2 pS).
Gabazine Inhibits mIPSCs but not Tonic Current.
We next tested
a series of GABAA receptor antagonists to
determine whether the tonic and synaptic currents could be
distinguished pharmacologically. Notably, the classical
GABAA receptor antagonists, bicuculline and
gabazine, had similar effects on mIPSCs but different effects on the
tonic current. Bicuculline abolished the mIPSCs and evoked a large
outward shift in the holding current. In contrast, the high-affinity
antagonist gabazine (1 µM) produced no significant shift in the
holding current; nonetheless, it completely abolished the mIPSCs (Fig.
2A) (n = 12 cells). These
observations suggest that the tonic current does not result from the
simple summation of unresolved mIPSCs. Gabazine has a higher affinity
for GABAA receptors than bicuculline. However,
despite this high affinity high concentrations of gabazine (10-20
µM) did not inhibit the tonic current (Fig. 2B). Analysis of the
tonic noise recorded during the application of gabazine (10 µM)
revealed that the unitary conductance of the underlying channels was
~4.3 pS (n = 15 cells), comparable with the channels
responsible for the tonic current recorded in the absence of gabazine
(see above).
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Gabazine Effects on Tonic Current in Rat Hippocampal Brain
Slice.
The complement of GABAA receptor
subunits changes with cell maturation and tissue culture conditions
(Laurie et al., 1992). Consequently, the apparent lack of effect of
gabazine on the tonic current might occur only in immature hippocampal
neurons grown in dissociated culture. To determine whether the tonic
current was evident in postnatal hippocampal neurons, we next recorded from the hippocampal slice preparation.
). As in
cultured neurons, applications of gabazine (20 µM) abolished mIPSCs
while causing only a slight, outward shift of the baseline tonic
current (3.5 ± 1.7 pA, P > 0.05). Whole-cell
currents were also recorded from acutely isolated neurons obtained from
the hippocampal slice to rule out the possibility that the tonic
current in postnatal hippocampal neurons was caused by spontaneous
opening of GABAA channels (Birnir et al., 2000).
This preparation provides excellent concentration-clamp conditions that
eliminate the exposure to neurotransmitter released from neighboring
cells. Both gabazine (1 µM-1 mM) and bicuculline (10 µM) failed to
activate a current in isolated neurons (data not shown) suggesting that
spontaneous channel openings did not account for the tonic current. In
addition, the lack of response to gabazine again indicates that
gabazine does not act as a weak partial agonist.
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The Tonic Current Is Enhanced by Midazolam in Cultured
Neurons.
We next tested whether the tonic current evident in
cultured neurons was sensitive to a sedative-hypnotic benzodiazepine, as recently reported in granule cerebellar neurons (Leao et al., 2000).
Classical benzodiazepines, including midazolam, do not directly
activate native GABAA receptors in the absence of
GABA, but potentiate GABA-evoked channel opening by increasing agonist affinity (Lavoie and Twyman, 1996). The application of midazolam produced an inward current, as illustrated in Fig.
4A. Flumazenil, a specific benzodiazepine
antagonist at the GABAA receptor, produced no
effect when applied in the absence of midazolam but reversed the
baseline shift induced by midazolam (Fig. 4B). These results suggest
that ambient GABA activates tonic current.
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). Maximal enhancement of the GABAergic current was
observed with concentrations of midazolam within the nM range whereas
the enhancement was reduced at higher concentrations (>1 µM), as
described previously (Rogers et al., 1994). The increase in the tonic
current by midazolam was blocked by bicuculline (Fig. 4A).
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; Bai et al.,
1999). We reasoned that if the tonic current results from persistent
low concentrations of GABA, then a fraction of this population would
desensitize and thus be enhanced by compounds that reduce
desensitization. It was predicted that low concentrations of propofol
that reduce desensitization but do not directly activate the receptor
would produce a greater increase in the tonic current compared with
benzodiazepines that do not reduce desensitization but simply increase
the apparent affinity for GABA. Applications of propofol induced a
shift in the baseline tonic current (Fig. 5, C and D) and the tonic
inward current increased in amplitude with increasing concentrations of
propofol. Unlike midazolam, the response to propofol did not saturate
but continued to increase with concentrations over the range tested
(0.2-5 µM), as described previously (Orser et al., 1994).
The concentration-response relationships for enhancement in the tonic
current by midazolam or propofol are summarized in Fig. 5B, D. Propofol, compared with midazolam, had a lower potency but higher
efficacy for increasing the amplitude of the tonic inward current.
Unlike midazolam, gabazine (1 µM) inhibited responses activated by 1 µM propofol by ~30% (70 ± 8% residual current; n = 6; P < 0.05). This is consistent
with the partial inhibition by gabazine of currents activated by the
anesthetics, pentobarbital and alphaxalone (Uchida et al., 1996; Ueno
et al., 1997).
Comparison of the Relative Increase in the Tonic Current and IPSCs
Caused by Midazolam and Propofol in Cultured Neurons.
Compounds
that reduce desensitization should enhance the tonic current more than
compounds that simply slow dissociation of the agonist. To highlight
the influence on deactivation and desensitization, we next compared the
effects of midazolam and propofol on the charge transfer associated
with the tonic and quantal postsynaptic currents. Changes in quantal
charge transfer associated with the mIPSCs are dominated by alterations
in the dissociation rate of agonist (Bai et al., 1999). In contrast,
changes in deactivation as well as desensitization rates of the
receptor should influence the charge transfer associated with the tonic current.
; Poncer et al., 1996; Rovira and Ben-Ari, 1999)
and propofol (Orser et al., 1994) produced a concentration-dependent
prolongation in the duration of mIPSCs (Fig.
6A). The threshold concentrations of
midazolam and propofol that increased the decay time of mIPSCs were
approximately 0.04 µM and 0.2 µM, respectively (Fig. 6A, Table
1). Discrete mIPSCs could be clearly
resolved even in the presence of saturating concentrations of midazolam
(0.2 µM). In contrast, when propofol was applied at concentrations
equal to or greater than 5 µM, the baseline noise and tonic current
increased such that mIPSCs could no longer be clearly resolved. Table 1
summarizes the changes in the amplitude and time course of the mIPSCs
caused by the various concentrations of midazolam and propofol and
Table 2 summarizes the effects of these
drugs on the frequency of mIPSCs. In addition to slowing the time
course of current decay, higher concentration of midazolam, and
intermediate concentrations of propofol, increased the frequency of the
mIPSCs. This effect of midazolam was not reported for mIPSCs investigated in the CA3 region of the hippocampal slice culture preparation (Poncer et al., 1996).
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Qdrug/Qcontrol)
produced by the various concentrations of midazolam and propofol. A
simple qualitative comparison indicated that both drugs caused a
greater increase in the absolute charge transfer associated with the
tonic current compared with mIPSCs (Fig. 6C). For example, midazolam
(0.2 µM) or propofol (1 µM) produced a 21- or 33-fold greater
increase in the absolute charge transfer, respectively, for the tonic
current compared with the mIPSCs (P < 0.05). Although
the absolute increase in the charge transfer is greater for the tonic
current, this is caused in part by the high baseline tonic current.
Therefore, the relative changes in the synaptic and tonic current
produced by the various concentrations of midazolam and propofol were
also examined as illustrated in Fig. 6D.
The above results describe the change in charge transfer associated
with miniature synaptic currents recorded in the presence of TTX.
Because the amplitude, frequency, and duration of action potential-dependent spontaneous IPSCs may be greater than those of
mIPSCs (Otis et al., 1991), we also compared the effects of midazolam
and propofol on the tonic current and synaptic currents recorded in the
absence of TTX. The peak amplitude (45 ± 5 pA, n = 8 cells) and area of spontaneous IPSC (1243 ± 156 ms × pA, n = 8) were similar to amplitude and
area of miniature IPSC (40 ± 2 pA and 1243 ± 80 ms × pA, n = 38, P = 0.99, respectively). However, the frequency of spontaneous IPSCs (1.4 ± 0.3 Hz, n = 8, P < 0.05) was increased
(by 2.3-fold). Despite the higher frequency of spontaneous IPSCs, the
increase in charge transfer associated with tonic currents by midazolam
and propofol was, nevertheless, still considerably more than that
associated with the synaptic current. Consistent with our previous
results, midazolam (0.04 µM) and propofol (0.2 µM) caused a 11-fold
(n = 4) and 32-fold (n = 4)
(P < 0.05) greater increase, respectively, in the
charge transfer mediated by the tonic current compared with the
spontaneous IPSCs.
Midazolam and Propofol Interact to Cause a Supra-Additive Increase
in the Tonic Current.
To further define the conditions of
GABAA receptor activation that underlie the tonic
current, we investigated the interaction between midazolam and
propofol. Isobolographic analysis indicated that midazolam and propofol
interact synergistically to increase GABAA
receptor function when receptors are activated by low (<3 µM) but
not high concentrations of GABA (McAdam et al., 1998). In contrast, the
interaction between these drugs is nonsynergistic when receptors are
activated by higher or near-saturating concentrations of GABA. We
reasoned that if the tonic current were activated by a low
concentration of GABA, then the combination of midazolam and propofol
would produce an effect greater than the predicted sum of the effects
of each drug alone. We observed that midazolam (40 nM) and propofol (1 µM) caused a supra-additive increase in the tonic current that was
greater than that predicted from linear summation (Fig.
7). When the benzodiazepine antagonist,
flumazenil, was applied together with propofol and midazolam, the
current returned to the amplitude observed when propofol was applied in the absence of midazolam. These results support the suggestion that the
tonic current is activated by a low ambient concentration of
transmitter.
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The Tonic and Synaptic Currents Could Be Mediated by a Distinct
Population of Receptors.
The differential pharmacological
sensitivity of the synaptic and tonic currents to gabazine could be
explained in one of two ways. Firstly, the subunit composition of a
distinct population of receptors could render them particularly
sensitive to background GABA levels such that they generate the tonic
current (Brickley et al., 1996). Alternatively, the receptors
underlying the tonic and synaptic current could contain a similar
structural complement of subunits, and different states of
the receptor account for the differential pharmacological sensitivity.
Tonic current may be activated by low persistent concentrations of GABA
whereas transient saturating concentrations of GABA activate mIPSCs.
Thus, either structural or pharmacodynamic factors could contribute to
the different sensitivity of the tonic and synaptic current to
gabazine, midazolam, and propofol. Kinetic modeling and computer simulation was used to further explore the characteristics of the tonic
current and account for the experimental findings. The apparent lack of
competition between gabazine and GABA in receptors responsible for the
tonic current led us to examine an allosteric model of gabazine
inhibition. The single channel conductance of the tonic channels was
estimated to be lower than that of the synaptic receptors activated
during mIPSCs. Because low concentrations of exogenous GABA also
elicited currents with a low single-channel conductance, we also
considered the possibility that monoliganded GABAA receptors open to a low conductance state.
The detailed model used here is not the only explanation for our
results but accounts for our findings.
). The model was designed to
minimize the number of states, whereas preserving some of the
complexity of the system. The rate constants in the scheme, under both
control and propofol conditions, are provided in Table
3. In the model presented here, the
mono-liganded state was allowed to open to a low conductance state
(25% of the doubly liganded state). The background concentration of
GABA was selected to produce a response that was 0.4% of the maximal
current. At this concentration, the GABA response was primarily
attributable to low conductance, mono-liganded receptors, and <10% of
the available receptors were in the slow desensitization
state.
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concluded that propofol slowed many of the rate
constants of the reaction scheme, including the rate of agonist
dissociation and the rate of entry into the two desensitized states.
This model predicts that propofol causes a greater increase in charge
transfer for the tonic current compared with mIPSCs (2 fold versus 1.5 fold, respectively). For the receptors underlying the tonic current,
bicuculline was assumed to bind to the GABAA receptor and prevent both GABA and gabazine binding. However, because
gabazine did not interfere with activation of the tonic receptor by
GABA, it was assumed to interfere with bicuculline binding by an
allosteric mechanism. In Scheme 1, all receptor states bind gabazine
equally well except for the bicuculline-bound state (BC), which
excludes gabazine binding. For clarity, the parallel set of gabazine
bound states are not shown. In this model, the addition of a high dose
of bicuculline (10 µM) causes a substantial inhibition of the tonic
current, as shown experimentally. The addition of 10 µM or 1 µM
gabazine has no effect on the tonic current. When the concentration of
bicuculline is increased 10-fold to 100 µM, bicuculline could
overcome the reciprocal allosteric effect of gabazine to compete with
GABA and reduce the tonic current. In Fig.
8B, we show the concentration-response
relationship predicted for gabazine reversal of bicuculline blockade
and its rightward shift caused by increasing bicuculline
concentrations. The experimental observations are superimposed on the
simulated curves (Fig. 8B, 
). Note the good agreement between the
electrophysiological data and predictions of the reciprocal allosteric
competition model.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The principal findings of this study are that the tonic and quantal synaptic currents exhibit distinct pharmacological sensitivities to gabazine and bicuculline as well as to two therapeutically important neurodepressive drugs. Simulation studies indicate that our electrophysiological data are consistent with the tonic being mediated by a population of receptors that bind gabazine in a manner that does not prevent channel opening by GABA. Most importantly, both midazolam and propofol evoked a greater increase in the total charge transfer of the tonic current compared with that associated with the prolongation of synaptic currents. These findings suggest a potential therapeutic role for the population of receptors responsible for the tonic current. Furthermore, we speculate that differences in the effects of midazolam and propofol on the tonic current may account for the differences between sedative and anesthetic compounds that act at the GABAA receptor.
The tonic current recorded here was insensitive to gabazine (SR-95531),
an aryl-aminopyridazine derivative that selectively binds to low
affinity GABAA receptors (Bureau and Olsen,
1990). Gabazine and bicuculline are generally considered to act as
competitive antagonists of the GABAA receptor
(Hamann et al., 1988; Ueno et al., 1997). However, gabazine and
bicuculline may not have identical mechanisms of action. Bicuculline
inhibits currents induced by both GABA and pentobarbital, whereas
gabazine does not antagonize current activated by pentobarbital in rat
hippocampal neurons (Uchida et al., 1996). Consistent with the notion
of distinct receptor populations, gabazine binding was shown previously
to coincide with the benzodiazepine 2 site, whereas bicuculline
colocalized with muscimol-preferring high-affinity sites (Olsen et al.,
1990).
Noise analysis indicated that "low conductance" channels mediated
the tonic current. A low unitary conductance ( = 6 pS) was also
evident in single channel recordings of GABAA
receptors from rat hippocampal neurons (Eghbali et al., 1997), and
neurons from the rat substantia nigra (Guyon et al., 1999). This
unitary conductance is lower than that reported for receptors that
mediate quantal synaptic currents in hippocampal neurons (~24-28 pS)
(De Koninck and Mody, 1994; Otis et al., 1994) and is lower than the main conductance of GABAA receptors studied using
single-channel recording methods (Orser et al., 1994). The low
conductance state may represent a mono-liganded form of the
GABAA receptor that predominates when receptors
are activated by low concentrations of ligand. Low conductance states
activated by low agonist concentrations have been reported for other
ligand-gated channels (Smith and Howe, 2000) although direct evidence
for concentration-dependent substate gating of
GABAA receptors is lacking at this time.
The source of GABA that activates the tonic current in culture and
slice is not known. The tonic current could be mediated by synaptic
receptors that are distant from the vesicular release sites and hence
exposed to subsaturating concentrations of transmitter (Mody et al.,
1994). Alternatively, spillover of vesicular released GABA could
activate receptors located extra-synaptically or at other synapses at
which quantal release has not occurred. It remains to be determined
whether the receptors underlying the tonic current in hippocampal
neurons are localized to synaptic and/or extra-synaptic regions of the
cells. Regardless of location, the differential responsiveness to
nonsaturating and saturating agonist concentrations could lead to
differential contributions of the tonic and quantal responses to
neurodepressive compounds.
The tonic conductance is not a phenomenon unique to immature neurons.
Persistent GABAergic currents have been recorded in the rat slice
preparation of postnatal and adult hippocampus (Otis et al., 1991);
cortex (Salin and Prince, 1996); and cerebellum (Brickley et al., 1996;
Wall and Usowicz, 1997). Furthermore, the relative importance of the
tonic current compared with synaptic currents may increase with
neuronal maturation. Age-dependent changes in the relative importance
of the tonic current and mIPSCs have been reported in postnatal granule
cells from rat cerebellum. The magnitude of the tonic current increased
during postnatal maturation, as did the ratio of charge transfer from
the tonic current compared with mIPSCs (Brickley et al., 1996).
Potentiation of Tonic Current and Synaptic Currents by Midazolam
and Propofol.
GABAA receptors activated by
persistent low concentrations of GABA are not subject to the same
strict temporal and spatial constraints as postsynaptic receptors
activated by vesicle-mediated quantal release. Although the amplitude
of the tonic current is much less than evoked synaptic currents, the
persistence of the tonic current results in a substantial integrated
charge transfer. As mentioned above, pharmacological modification of
GABAA receptors depends on the occupancy of the
receptor by GABA and the state of receptor activation and
desensitization. The greatest increase in GABAA
receptor activity produced by benzodiazepines and anesthetics occurs
when receptors are activated by low concentrations of transmitter (Harris et al., 1995). Consequently, GABAA
receptors activated by a low concentration of GABA are likely to be
more sensitive to benzodiazepines and anesthetics. Indeed,
benzodiazepines and anesthetics caused a relatively greater enhancement
of the tonic current compared with synaptic when measured as an
absolute increase in charge transfer (Fig. 6C). It is generally assumed
that the binding of GABA to the postsynaptic receptor is
diffusion-limited, with the peak of the IPSC occurring when the free
concentration of GABA is high. Factors that increase agonist binding
are not expected to influence the peak amplitude. Accordingly,
midazolam and propofol generally exerted little effect on the amplitude of mIPSCs, but instead prolonged their duration. The decay of IPSCs
probably occurs during or after the clearance of GABA from the cleft
(De Koninck and Mody, 1994). Thus, gating steps and the unbinding of
GABA regulate the time course of IPSCs. Presumably, the prolongation of
mIPSCs by midazolam and propofol results from a reduction in agonist dissociation.
Charge Transfer Mediated by IPSCs Compared with the Tonic Current: Clinical Implications. Although acknowledging that general anesthetics and benzodiazepines influence a variety of neuronal receptors, overwhelming evidence has implicated the GABAA receptor as a primary target. A major neurodepressive action of benzodiazepines and anesthetics may be to enhance a tonic GABAergic inhibition as well as prolong synaptic currents. The concentrations of midazolam and propofol used in our experiments are similar to the free concentrations in the plasma, measured in patients during anesthesia. We compared the relative efficacy of propofol and midazolam in increasing the tonic current and low concentrations of propofol (>1 µM) activated a greater increase in the tonic current compared with that of saturating concentrations of midazolam.
The relative efficacy of propofol and midazolam to enhance the tonic current, but not synaptic currents, seems to be consistent with important differences in the clinical efficacy of anesthetics and benzodiazepines. Both propofol and midazolam obtund memory and consciousness but only propofol produces a level of neurodepression sufficient to prevent movement in response to painful stimuli. Propofol has a narrow therapeutic index and causes respiratory arrest when administered in excessive doses. In contrast, an overdose of midazolam or diazepam is rarely fatal, suggesting a "ceiling effect". The "ceiling effect" with midazolam but not propofol is also observed for electroencephalogram waveform changes. Finally, propofol is effective for the treatment of status epilepticus that is refractory to diazepam or midazolam. The extracellular concentration of GABA is reduced in epileptic hippocampi (During et al., 1995) and under these
conditions propofol may act as a surrogate agonist and activate a
profound increase in tonic inhibition. We speculate that
benzodiazepines are comparably less effective because they serve only
to potentiate the tonic current, which is abnormally reduced because of
the low concentrations of ambient GABA.
In summary, the GABAA receptors underlying the
tonic current are distinct from those activated during the generation
of mIPSCs and quantal synaptic transmission. The tonic channels may
serve as a novel target for benzodiazepines and anesthetic drugs and we
speculate that an increase in the tonic current contributes to the
clinical properties of these drugs.
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Footnotes |
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Received August 9, 2000; Accepted December 22, 2000
Send reprint requests to: Dr. B.A. Orser, Department of Physiology, Medical Science Building, Room 3318, University of Toronto, 1 King's College Circle, Toronto, Ontario, CANADA, M5S 1A8. E-mail: beverley.orser{at}utoronto.ca
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Abbreviations |
|---|
GABA, -aminobutyric acid;
IPSC, inhibitory
postsynaptic currents;
mIPSC, miniature inhibitory postsynaptic
current;
aCSF, artificial cerebrospinal fluid;
TTX, tetrodotoxin.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-aminobutyric acid-A receptor protein show different ligand-binding affinities.
Mol Pharmacol
37:
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V. B. Caraiscos, E. M. Elliott, K. E. You-Ten, V. Y. Cheng, D. Belelli, J. G. Newell, M. F. Jackson, J. J. Lambert, T. W. Rosahl, K. A. Wafford, et al. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by {alpha}5 subunit-containing {gamma}-aminobutyric acid type A receptors PNAS, March 9, 2004; 101(10): 3662 - 3667. [Abstract] [Full Text] [PDF]
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N. I. Kononenko and F. E. Dudek Mechanism of Irregular Firing of Suprachiasmatic Nucleus Neurons in Rat Hypothalamic Slices J Neurophysiol, January 1, 2004; 91(1): 267 - 273. [Abstract] [Full Text] [PDF]
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M. T. Bianchi and R. L. Macdonald Neurosteroids Shift Partial Agonist Activation of GABAA Receptor Channels from Low- to High-Efficacy Gating Patterns J. Neurosci., November 26, 2003; 23(34): 10934 - 10943. [Abstract] [Full Text] [PDF]
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A. J. Woodall, H. Naruo, D. J. Prince, Z. P. Feng, W. Winlow, M. Takasaki, and N. I. Syed Anesthetic Treatment Blocks Synaptogenesis But Not Neuronal Regeneration of Cultured Lymnaea Neurons J Neurophysiol, October 1, 2003; 90(4): 2232 - 2239. [Abstract] [Full Text] [PDF]
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K. Jensen, C.-S. Chiu, I. Sokolova, H. A. Lester, and I. Mody GABA Transporter-1 (GAT1)-Deficient Mice: Differential Tonic Activation of GABAA Versus GABAB Receptors in the Hippocampus J Neurophysiol, October 1, 2003; 90(4): 2690 - 2701. [Abstract] [Full Text] [PDF]
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G. B. Richerson and Y. Wu Dynamic Equilibrium of Neurotransmitter Transporters: Not Just for Reuptake Anymore J Neurophysiol, September 1, 2003; 90(3): 1363 - 1374. [Abstract] [Full Text] [PDF]
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F.-C. Hsu, R. Waldeck, D. S. Faber, and S. S. Smith Neurosteroid Effects on GABAergic Synaptic Plasticity in Hippocampus J Neurophysiol, April 1, 2003; 89(4): 1929 - 1940. [Abstract] [Full Text] [PDF]
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Y. Wu, W. Wang, and G. B. Richerson Vigabatrin Induces Tonic Inhibition Via GABA Transporter Reversal Without Increasing Vesicular GABA Release J Neurophysiol, April 1, 2003; 89(4): 2021 - 2034. [Abstract] [Full Text] [PDF]
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A. Bacci, J. R. Huguenard, and D. A. Prince Functional Autaptic Neurotransmission in Fast-Spiking Interneurons: A Novel Form of Feedback Inhibition in the Neocortex J. Neurosci., February 1, 2003; 23(3): 859 - 866. [Abstract] [Full Text] [PDF]
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J. Y. T. Yeung, K. J. Canning, G. Zhu, P. Pennefather, J. F. MacDonald, and B. A. Orser Tonically Activated GABAA Receptors in Hippocampal Neurons Are High-Affinity, Low-Conductance Sensors for Extracellular GABA Mol. Pharmacol., January 1, 2003; 63(1): 2 - 8. [Abstract] [Full Text] [PDF]
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P. M. Baker, P. S. Pennefather, B. A. Orser, and F. K. Skinner Disruption of Coherent Oscillations in Inhibitory Networks With Anesthetics: Role of GABAA Receptor Desensitization J Neurophysiol, November 1, 2002; 88(5): 2821 - 2833. [Abstract] [Full Text] [PDF]
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Z. Nusser and I. Mody Selective Modulation of Tonic and Phasic Inhibitions in Dentate Gyrus Granule Cells J Neurophysiol, May 1, 2002; 87(5): 2624 - 2628. [Abstract] [Full Text] [PDF]
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S. Mennerick, C.-M. Zeng, A. Benz, W. Shen, Y. Izumi, A. S. Evers, D. F. Covey, and C. F. Zorumski Effects on gamma -Aminobutyric Acid (GABA)A Receptors of a Neuroactive Steroid That Negatively Modulates Glutamate Neurotransmission and Augments GABA Neurotransmission Mol. Pharmacol., October 1, 2001; 60(4): 732 - 741. [Abstract] [Full Text] [PDF]
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L. S. Overstreet and G. L. Westbrook Paradoxical Reduction of Synaptic Inhibition by Vigabatrin J Neurophysiol, August 1, 2001; 86(2): 596 - 603. [Abstract] [Full Text] [PDF]
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1 µM). The EC50 was 28 nM, Imax was 23 pA, and
nH = 1.2. C, propofol caused a
dose-dependent increase in the tonic current. The tonic current
produced by propofol (Prop, 5 µM) was reversibly blocked by
bicuculline (10 µM, bottom trace). D, the concentration-response
relationship for the tonic current recorded in the presence of propofol
is shown. Each point represents the average values (±S.E.M.) for
currents recorded from 5 to 7 different cells. The dotted line
indicates that higher concentrations of propofol produce an even
greater increase in the current, as we previously reported (Orser et
al., 1994
) are shown (left). Midazolam produced a 7- to 21-fold greater
increase in charge transfer for the tonic current compared with mIPSCs
(P < 0.05). Similar to midazolam, propofol
produced a 6- to 33-fold greater increase in charge transfer associated
with the tonic current compared with mIPSCs (right). D, the relative
charge transfer associated with mIPSCs (left) and tonic current (right)
for propofol (
