Introduction

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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 GABA_A 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 GABA_A 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 GABA_A receptors (Lerma et al., 1986). Alternatively, the tonic current might result from spontaneous openings of constitutively active GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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).

	Materials and Methods

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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.

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.

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.

	Results

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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 GABA_A receptors (Valeyev et al., 1993) because it was also inhibited by another GABA_A 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).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Tonic and synaptic GABAergic currents in cultured hippocampal neurons under different experimental conditions. A, the upper trace illustrates currents recorded from cultured neurons in the absence or presence of bicuculline (BIC, 10 µM). Bicuculline abolished mIPSCs and induced an outward shift of the holding current (20 pA). The dashed line depicts the holding current in the absence of bicuculline. The lower traces are temporal expansions of two short segments. B, the unitary conductance of channels underlying the tonic current was estimated using variance analysis. The segments of the records that contained miniature IPSCs were removed to calculate the current variance. The amplitude of the current (Imean) was measured as the difference in the holding current measured before and after the application of bicuculline as indicated in panel 1C. Data were obtained from 17 neurons and the variance (pA2) value was plotted against the amplitude to the bicuculline-sensitive current. The solid line is a linear regression fit to all the data points. The estimated conductance was ~5.6 pS. C, inward current was activated by the application of low concentrations of GABA (0.1, 0.3 and 1 µM) applied to the neurons. The arrow indicates a mIPSC evident under control conditions. The variance was plotted against the mean current amplitude. Data were obtained from seven different neurons and the unitary conductance was estimated to be ~6.2 pS.

Gabazine Inhibits mIPSCs but not Tonic Current. We next tested a series of GABA_A receptor antagonists to determine whether the tonic and synaptic currents could be distinguished pharmacologically. Notably, the classical GABA_A 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 GABA_A 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).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Gabazine blocks mIPSCs but not the tonic current and reduces the inhibition by bicuculline. A, the tonic current was sensitive to bicuculline (10 µM) but not inhibited by gabazine (1 µM). Gabazine (1 or 10 µM) when applied alone did not cause a significant shift in the baseline current but abolished the mIPSCs. In contrast, bicuculline abolished the mIPSCs and caused an outward shift in the baseline. Current traces were filtered at a high cut-off frequency of 100 Hz. B, coapplication of gabazine and bicuculline caused the baseline to shift less than that observed in the presence of bicuculline alone. C, the bar graph illustrates the change in current amplitude after the application of bicuculline (10 µM, n = 8) or gabazine (1 µM; n = 8; P < 0.05). The changes in the amplitude of the current when bicuculline (10 µM) was coapplied with gabazine 1 µM, (n = 5), 10 µM (n = 5) or 100 µM (n = 6) are also shown. Note that the inhibition of the tonic current was reduced when bicuculline coapplied with gabazine 1 and 10 µM (P < 0.05) but not when bicuculline was applied alone.

Gabazine Effects on Tonic Current in Rat Hippocampal Brain Slice. The complement of GABA_A 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.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   The effects of gabazine and bicuculline on the tonic and synaptic currents recorded in rat hippocampal slices. The application of bicuculline (10 µM) abolished the mIPSCs and consistently caused a decrease in the holding current as indicated by an outward shift in the baseline and decrease in the noise. In contrast, gabazine (20 µM) inhibited the transient synaptic currents but caused no outward shift, as indicated in the current traces and summarized in the bar graph.

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 GABA_A 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 GABA_A 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.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Midazolam increases the tonic current in the absence and presence of gabazine. A, the bicuculline-sensitive tonic current observed after the application of midazolam (bar). The dashed lines indicate the holding current under control conditions and the transient downward deflections represent IPSCs. B, flumazenil (10 µM) reversed the increase in the tonic current caused by midazolam. C, current traces from a single neuron illustrate that midazolam (40 nM) produced the same increase of the tonic current when applied in the absence or presence of gabazine (1 µM). Midazolam did not change the current amplitude in the presence of bicuculline. D, the effects of midazolam on the tonic current recorded under control conditions or in the presence of bicuculline and gabazine are summarized in the bar graph. Note that the values are compared with the amplitude of the midazolam-induced current. No significant decrease in the tonic current was observed in the presence of gabazine (1 µM), whereas bicuculline significantly reduced the effects of midazolam on tonic current (P < 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Propofol and midazolam cause a concentration-dependent increase in the amplitude of the tonic current. A and B, the concentration-response relationship for the tonic inward current, recorded in the presence of midazolam, is shown. Each data point represents the averaged values (±S.E.M.) obtained from 5 to 9 different cells. The smooth curve represents the data fit using a modified Hill equation (I = Imax/(1 + (C/EC50)nH) where Imax is the maximal response, nH is the Hill coefficient, and EC50 is the concentration that produced 50% of the maximal response (for concentrations  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).

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.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Midazolam and propofol produce a greater increased in the charge transfer associated with the tonic current compared with mIPSCs. A, the average of 100 to 150 individual mIPSCs is shown before and after an application of 0.2 µM midazolam (MDZ) or 1 µM propofol (Prop). The smooth solid lines indicate the fit of a single exponential function obtained using an iterative nonlinear Levenberg-Marquadt algorithm. The time constants () of the decay phase were consistently increased by midazolam (MDZ) and propofol (Prop). The peak amplitude of mIPSCs was not significantly increased by midazolam (0.08-1 µM) or propofol (0.04-0.2 µM; see Table 1). However, 1 µM propofol increased the peak current by 12 ± 4% (n = 7; P < 0.05). B, the schematic drawings and equations illustrate the methods used to calculate charge transfer per unit of time where QmIPSP is the increase in charge transfer associated with mIPSCs per second; fcon and fdrug are the frequencies of mIPSC under control conditions and during drug application; Qcon and Qdrug are the average values for charge transfer per mIPSC under control conditions and during drug applications, respectively; QTC is the increase in charge transfer associated with the tonic current (represented by the shaded area under the steady-state current amplitude). ITC represents the amplitude of the steady state current. C, the relationship of midazolam concentration and the charge transfers associated with mIPSCs () and with tonic current () 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 () and midazolam () are shown.

Midazolam and Propofol Interact to Cause a Supra-Additive Increase in the Tonic Current. To further define the conditions of GABA_A 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 GABA_A 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.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Midazolam and propofol cause a supra-additive increase in the tonic current. A, the increase in amplitude of the tonic current by propofol (1 µM), midazolam (40 nM), and propofol plus midazolam is shown. B, the bar graph summarizes the changes in amplitude of the tonic current. The peak current amplitudes were measured in the presence of propofol 1 µM (53 ± 8 pA; n = 12), midazolam 40 nM (11 ± 2 pA; n = 12), propofol plus midazolam (106 ± 12; n = 12) and propofol, midazolam, and flumazenil. The amplitude of the tonic current calculated (cal.) for an additive interaction between midazolam and propofol (64 ± 10; n = 12) was less than actual measured (real) response (P < 0.05).

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 GABA_A 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.

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View larger version (18K): [in this window] [in a new window]   Fig. 8.   Simulations of the effects of bicuculline and gabazine on the GABA-induced tonic and quantal synaptic currents. A, simulations of GABAAR-mediated activity generated by a tonic application of a low concentration of GABA. The persistent GABA signal generates a tonic current approximately 0.4% of the maximal possible response if all receptors were fully activated. Different combinations of bicuculline and gabazine concentrations on the tonic and synaptic currents, in the presence and absence of propofol were simulated. We selected a concentration of propofol that increased the area of the synaptic-like responses by 1.5-fold and increased the tonic current 2-fold. The dissociation constants of bicuculline and gabazine were set at 0.1 and 0.9 µM, respectively. Application of bicuculline (10 and 100 µM, solid bars), and gabazine (10 µM, open bar) are indicated. B, dose-response relations for the effects of gabazine on bicuculline inhibition of GABAA receptors in the presence of 0.8 µM GABA. At low gabazine concentrations (where GABA-occupied receptors dominate the current), 1 µM bicuculline reduces the tonic current somewhat, although 100 µM bicuculline causes a near-complete inhibition. , the experimentally measured values of tonic current in the presence of different combinations of gabazine and bicuculline. Note the good agreement between the electrophysiological data and the simulations that assumes reciprocal competitive allosteric interactions between bicuculline and gabazine.

	Discussion

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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 GABA_A receptor.

The tonic current recorded here was insensitive to gabazine (SR-95531), an aryl-aminopyridazine derivative that selectively binds to low affinity GABA_A receptors (Bureau and Olsen, 1990). Gabazine and bicuculline are generally considered to act as competitive antagonists of the GABA_A 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 GABA_A 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 GABA_A receptors studied using single-channel recording methods (Orser et al., 1994). The low conductance state may represent a mono-liganded form of the GABA_A 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 GABA_A 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. GABA_A 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 GABA_A receptors depends on the occupancy of the receptor by GABA and the state of receptor activation and desensitization. The greatest increase in GABA_A receptor activity produced by benzodiazepines and anesthetics occurs when receptors are activated by low concentrations of transmitter (Harris et al., 1995). Consequently, GABA_A 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 GABA_A 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.