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Benzodiazepines (BDZs) are known to increase the amplitude and duration of IPSCs. Moreover, at low [GABA], BDZs strongly enhance GABAergic currents suggesting the up-regulation of agonist binding while their action on gating remains a matter of debate. In the present study we have examined the impact of flurazepam and zolpidem on mIPSCs by investigating their effects on GABAAR binding and gating and by considering dynamic conditions of synaptic receptor activation. Flurazepam and zolpidem enhanced the amplitude and prolonged decay of mIPSCs. Both compounds strongly enhanced responses to low [GABA] but, surprisingly, decreased the currents evoked by saturating or half-saturating [GABA]. Analysis of current responses to ultrafast GABA applications indicated that these compounds enhanced binding and desensitization of GABAA receptors. Flurazepam and zolpidem markedly prolonged deactivation of responses to low [GABA] but had almost no effect on deactivation at saturating or half-saturating [GABA]. Moreover, at low [GABA], flurazepam enhanced desensitization–deactivation coupling but zolpidem did not. Recordings of responses to half-saturating [GABA] applications revealed that appropriate timing of agonist exposure was sufficient to reproduce either a decrease or enhancement of currents by flurazepam or zolpidem. Recordings of currents mediated by recombinant (‘synaptic’) α1β2γ2 receptors reproduced all major findings observed for neuronal GABAARs. We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.
GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the adult mammalian central nervous system. To date, as many as 20 subunits of GABAA receptors (γ-aminobutyric acid receptor type A) (α1–6, β1–4, γ1–3, δ, ρ1–3, ɛ, π and θ) have been cloned (Cherubini & Conti, 2001; Fritschy & Brunig, 2003) suggesting an overwhelming heterogeneity. However, most common GABAARs consist of two α, two β and one γ or δ subunit (Whiting, 2003; Wafford, 2005).
Benzodiazepine (BDZ) receptor agonists are known as positive modulators of specific GABAA receptors (GABAARs) (Rudolph & Mohler, 2004; Wafford, 2005; Rudolph & Mohler, 2006). BDZs were commonly found to enhance the amplitude and to prolong GABAergic IPSCs (inhibitory postsynaptic currents) (Frerking et al. 1995; Nusser et al. 1997; Perrais & Ropert, 1999; Hajos et al. 2000; Perrais & Ropert, 2000). Several lines of evidence indicate that BDZs up-regulate the binding affinity of GABAARs. A compelling indication for this mechanism is a strong BDZ-induced enhancement of amplitude and onset rate of currents evoked by non-saturating [GABA] but these effects tend to disappear at saturating [GABA] (Lavoie & Twyman, 1996; Krampfl et al. 1998). BDZs do not clearly affect the GABAAR single channel lifetimes (Twyman et al. 1989; Rogers et al. 1994). These findings, taken altogether, suggest that BDZs enhance binding affinity while their effect on GABAAR gating appears minor. However, Rusch & Forman (2005) as well as Downing et al. (2005) have proposed a novel mechanism for GABAAR modulation by BDZs. They considered a spontaneously active GABAAR mutant and reported that BDZs might effectively modulate the GABAAR gating. However, it was not clear to what extent these observations applied to the native GABAARs in which coupling between binding and gating has not been altered by mutations. More recently, Campo-Soria et al. (2006) have reported that in oocytes expressing ultrahigh levels of wild type GABAA receptors, a high concentration (1 μm) of diazepam induced a small but detectable GABAergic current and proposed that this BDZ might act on GABAARs by affecting the opening transitions. In a recent study, we have reported that flurazepam and zolpidem affected both binding and gating of α1β2γ2 receptors (Mercik et al. 2007). The effect on receptor gating was deduced from a BDZ-induced decrease in amplitude and a moderate modulation of the time course of currents elicited by saturating [GABA]. These observations appeared peculiar as they were substantially different from what is commonly observed for synaptic currents. It is thus interesting to investigate whether the effects of flurazepam and zolpidem observed for α1β2γ2 receptors can be reproduced in neurons. In particular, it is appealing to compare the action of these drugs on currents evoked by exogenous GABA and on mIPSCs (miniature inhibitory postsynaptic currents). For this purpose, the effect of flurazepam and zolpidem has been examined on mIPSCs and on current responses to ultrafast GABA applications recorded from rat cultured hippocampal neurons. We found that flurazepam and zolpidem modulated both binding and gating of neuronal receptors. However, BDZ effects on amplitudes of mIPSCs and on responses to saturating [GABA] showed qualitative differences. To explore this discrepancy, we have modelled the synaptic currents by responses to short applications of non-saturating [GABA] and concluded that extreme non-equilibrium conditions, dictated by a fast agonist transient, render mIPSCs particularly susceptible to modulation by BDZs.
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The major finding of the present work is that the non-equilibrium conditions of synaptic GABAAR activation, dictated by a very brief synaptic agonist transient (Clements, 1996; Mozrzymas et al. 1999, 2003b; Overstreet et al. 2002; Mozrzymas, 2004), have a crucial impact on IPSC sensitivity to BDZ receptor agonists. It was puzzling that mIPSCs were enhanced by flurazepam and zolpidem while responses evoked by saturating or half-saturating [GABA] were down-regulated by these drugs (Figs 1 and 3). This discrepancy could not result from different GABAAR subtypes in synapses and in excised patches (or whole cells) because a similar BDZ effect was observed for α1β2γ2 receptors (Fig. 8; Mercik et al. 2007) that are abundantly present in GABAergic synapses (e.g. Farrant & Nusser, 2005). Moreover, our experimental data (Figs 7 and 8) and model simulations (Fig. 9) show that for non-saturating [GABA], appropriate timing of GABA applications is sufficient to reproduce such apparently opposite BDZ effects. This reflects a general rule that a very brief agonist transient renders the IPSCs particularly sensitive to modulators affecting GABA binding and the larger the distance from saturation, the larger the impact of such modifiers (Mozrzymas et al. 1999, 2003b; Mozrzymas, 2004).
As explained above, the most parsimonious mechanism for observed effects of flurazepam and zolpidem is an up-regulation of binding and desensitization. Although modulation of GABAAR gating by both BDZ receptor agonists considered here appears most compatible with enhancement of desensitization, there were qualitative differences in modulation of gating by flurazepam and zolpidem. The impact of these drugs on deactivation–desensitization coupling (Fig. 5) and on desensitization (Fig. 6) was clearly different. Our data are insufficient to ascribe these differences to, for example, differential modulation of distinct desensitized conformations. Moreover, we cannot exclude that these compounds additionally affected conformational transitions other than desensitization (e.g. open/closed) but for reasons presented in ‘Model simulations’ we believe that their impact is minor. The mechanism proposed here differs from that recently suggested by Rusch & Forman (2005) and Downing et al. (2005), who postulated enhancement in efficacy by BDZs in a spontaneously active GABAAR mutant. However, mutations rendering the receptors spontaneously active could affect also their sensitivity to BDZs. More recently, however, Campo-Soria et al. (2006) have observed that in oocytes expressing ultrahigh levels of wild type GABAARs, diazepam induced a detectable current that was considered as further evidence for the enhancement of GABAAR efficacy by diazepam. Although our data are not sufficient to discriminate between these mechanisms, there are some points that are worth pointing out. A substantial enhancement of receptor efficacy by BDZs would be expected to result in a modification of open and/or closed time distributions that has not been observed in classical single channel studies (Twyman et al. 1989; Rogers et al. 1994). Since the peak of response depends on the occupancy balance of closed, open and desensitized states, it is expected that the BDZs-induced increase in efficacy would increase the amplitude of current evoked by saturating [GABA]. However, our experiments show the opposite (Fig. 3). While there is a general agreement that modulation of receptor desensitization may critically shape the time course of GABAergic currents, the impact of this conformation has not been considered in the papers suggesting BDZ-induced enhancement of efficacy. Finally, we have made an attempt to record currents evoked by up to 10 μm flurazepam or to 3 μm zolpidem but no detectable currents were observed (in some cultures GABAAR expression was quite high: whole-cell responses to 1 μm GABA were above 1 nA at −50 mV with current resolution of ∼5–10 pA). It is thus possible that diazepam might act differently than flurazepam or zolpidem. Taking altogether, in our view, the works discussed above (Rusch & Forman, 2005; Downing et al. 2005; Campo-Soria et al. 2006) raise an interesting possibility that BDZs might affect the receptor efficacy but do not exclude their effect on binding or desensitization and the precise contributions of these mechanisms remain to be assessed.
It is surprising that both flurazepam and zolpidem strongly slowed down the deactivation of currents evoked by low (1–30 μm) GABA (Fig. 4) while for responses to higher [GABA] this effect was not present (except for a subtle effect at 10 μm flurazepam). Importantly, this pattern was observed both for neuronal GABAARs and recombinant α1β2γ2 receptors (Figs 3 and 8). It is still more puzzling that the BDZ receptor agonists considered here prolonged mIPSCs (Fig. 2), while deactivation of currents elicited by 100 μm and higher [GABA] was not affected. Although we have no definite explanation for these discrepancies, we would like to propose a mechanism that appears plausible and is compatible with all our experimental findings. The fact that the major effects of BDZs on amplitude and on deactivation kinetics are observed at GABA concentration markedly lower than the EC50 value, suggests that they require conditions in which a considerable percentage of receptors are singly bound. Macdonald et al. (1989) have proposed that, at micromolar [GABA], a considerable proportion of channel activity is due to singly bound GABAA receptors. The increase in binding affinity by BDZ would increase the percentage of doubly bound receptors. This, in turn, would alter the deactivation kinetics because the rate constants of opening/closing and desensitization are different in doubly and singly bound receptors. This prediction is, to a smaller or larger extent, reproduced by each model in which singly bound open states are postulated (e.g. Macdonald et al. 1989; Jones & Westbrook, 1995). Such a mechanism predicts that at higher [GABA], at which fully bound GABAARs are predominant, BDZ impact on deactivation would be smaller. However, a clear effect of BDZs on mIPSCs decay kinetics (Fig. 2) remains puzzling, as synaptic [GABA] certainly exceeds micromolar concentrations. Assuming that the synaptic agonist efficiency is reasonably described by the product of peak concentration and duration of agonist exposure (Barberis et al. 2004), the action of synaptic GABA transient would be comparable to application of hundreds of micromoles for ∼1 ms. However, our experiments show that at 100 μm GABA, BDZs did not affect deactivation either for neuronal or recombinant α1β2γ2 receptors. An alternative possibility is that synaptically released agonist spills over from the cleft and activates perisynaptic GABAARs. Clearly, GABA spilling over from a synapse reaches perisynaptic receptors at concentrations lower than in the cleft and therefore the ensuing current is more susceptible to modulation by BDZs. The enhancement of this current would appear as an up-regulation of the ‘slow’ IPSC component because of prolonged diffusion and slower kinetics due to low [GABA]. A similar proposal that IPSCs are partially shaped by subsaturating [GABA] has been put forward by Hill et al. (1998) who studied modulation of GABAergic synaptic currents by diethyl-lactam that affected currents evoked by non-saturating [GABA] but had no effect on responses to saturating [GABA]. There is a general agreement that the phenomenon of GABA spill-over may have a pronounced impact on GABAergic currents, especially in the case of intense synaptic activity (e.g. Isaacson et al. 1993; Overstreet & Westbrook, 2003) although blockade of GABA uptake exerts only a weak, if any, effect on mIPSCs. Interestingly, Hill reported that the contribution of subsaturating [GABA] to IPSCs was insensitive to blockade of the GABA uptake system. Altogether, we propose that the major mechanisms of mIPSCs modulation by BDZs are related to: (i) enhancement of the binding rate that has a particularly strong effect in conditions of non-saturation and fast agonist transient, and (ii) enhancement of current evoked by low [GABA] spilling over from the synapse that is manifested as a slow down of mIPSC. Although the latter proposal is compatible with our observations, we have no direct evidence for it and therefore, at the present stage, it remains speculative. Moreover, kinetic behaviour of synaptic and extrasynaptic GABAA receptors (even of the same type) might differ because of possible modulatory post-translational processes mediated by, for example, phosphorylation/dephosphorylation or binding to regulatory proteins (e.g. GABARAP or gephyrine).
Contrary to our findings, Mellor & Randall (1997) and Krampfl et al. (1998) reported that BDZs prolonged deactivation kinetics of currents evoked by high [GABA]. The reason for this discrepancy is not clear. However, in these studies the fast deactivation component was at least one order of magnitude slower than that reported here. This may suggest that in the protocols applied by Mellor & Randall (1997) and Krampfl et al. (1998) the fast component was not detected. Other factors, such as differences in benzodiazepine type, cellular model or GABA application protocols could also underlie these different observations.
The observation that BDZ receptor agonists might affect different GABAAR properties (binding and gating) is not surprising. Walters et al. (2000) provided evidence for two functionally distinct BDZ binding sites on α1β2γ2 receptors. It is thus possible that different sensitivities to flurazepam of currents evoked by 1 μm GABA (Fig. 3) and on deactivation of currents evoked by saturating [GABA] (a significant effect only at 10 μm, Fig. 4) might involve the presence of different BDZ binding sites.
An important conclusion of this work is that the proposed mechanisms of BDZ receptor agonist action implicates them as potent modulators of both tonic and phasic GABAergic currents. Qualitatively, their effect on the tonic component appears larger, which seems important as it is believed that tonic currents mediate considerably larger charge transfer than the phasic ones (Farrant & Nusser, 2005). On the other hand, it needs to be born in mind that the tonic and phasic forms of inhibition are functionally coupled. Indeed, ambient [GABA] depends on network excitability while shunting (tonic) GABAergic conductance has a major impact on the neuronal firing and therefore on synaptic signalling.