Limits...
GABA transient sets the susceptibility of mIPSCs to modulation by benzodiazepine receptor agonists in rat hippocampal neurons.

Mozrzymas JW, Wójtowicz T, Piast M, Lebida K, Wyrembek P, Mercik K - J. Physiol. (Lond.) (2007)

Bottom Line: 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.We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroscience, Department of Biophysics, Wrocław Medical University, Chałubiñskiego 3, 50-368 Wroclaw, Poland.

ABSTRACT
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 GABA(A)R 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 GABA(A) 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') alpha1beta2gamma2 receptors reproduced all major findings observed for neuronal GABA(A)Rs. We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.

Show MeSH

Related in: MedlinePlus

Flurazepam and zolpidem differentially affect the deactivation–desensitization coupling A, B and C, examples of current traces evoked by 1 μm, 30 μm and 10 mm GABA, respectively, applied for short (thin line) and long time (thick line). The following durations of agonist applications were used at different [GABA]: 1 μm, 2 s; 3 μm, 1 s; 10 μm, 50 ms; 30 μm, 10 ms; 100 μm, 5 ms; 10 mm, 1 ms and the respective long applications were five times longer (see text for explanation of how short and long application times were defined). Right panels in A, B and C show the same current traces but normalized to the current value at the end of GABA pulse. In C, the inset shows the rapid phases of current responses on an expanded time scale. Note that at high [GABA] the deactivation following brief GABA pulse is much faster than that after a long application (mainly due to a predominant rapid deactivation component in the former one). D, the ratio of τmean values measured for deactivation currents following long and brief GABA applications, respectively, versus GABA concentration. Note that pulse duration affects τmean only for GABA concentrations equal or above 30 μm at which rapid desensitization becomes prominent. E, ratio of time constants of slow deactivation components (τslow) measured following long and brief GABA applications, respectively, versus[GABA]. In D and E, mean values were calculated from at least n = 8 cells. F, relative τmean for deactivation currents in the presence of 3 μm of flurazepam (with respect to control conditions) for short pulses (black bars) and for long pulses (middle hatched bars). Cross-hatched bars show the ratios of relative τmean values measured for long and short pulses, respectively. G, analogous results as presented in F but obtained for 3 μm of zolpidem. In F and G, mean values were calculated from at least n = 5 cells. Note that at low [GABA] flurazepam enhances the deactivation–desensitization coupling and zolpidem does not. Insets above current traces depict the time course of applied agonist. *Significant difference with respect to the control values.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2375456&req=5

fig05: Flurazepam and zolpidem differentially affect the deactivation–desensitization coupling A, B and C, examples of current traces evoked by 1 μm, 30 μm and 10 mm GABA, respectively, applied for short (thin line) and long time (thick line). The following durations of agonist applications were used at different [GABA]: 1 μm, 2 s; 3 μm, 1 s; 10 μm, 50 ms; 30 μm, 10 ms; 100 μm, 5 ms; 10 mm, 1 ms and the respective long applications were five times longer (see text for explanation of how short and long application times were defined). Right panels in A, B and C show the same current traces but normalized to the current value at the end of GABA pulse. In C, the inset shows the rapid phases of current responses on an expanded time scale. Note that at high [GABA] the deactivation following brief GABA pulse is much faster than that after a long application (mainly due to a predominant rapid deactivation component in the former one). D, the ratio of τmean values measured for deactivation currents following long and brief GABA applications, respectively, versus GABA concentration. Note that pulse duration affects τmean only for GABA concentrations equal or above 30 μm at which rapid desensitization becomes prominent. E, ratio of time constants of slow deactivation components (τslow) measured following long and brief GABA applications, respectively, versus[GABA]. In D and E, mean values were calculated from at least n = 8 cells. F, relative τmean for deactivation currents in the presence of 3 μm of flurazepam (with respect to control conditions) for short pulses (black bars) and for long pulses (middle hatched bars). Cross-hatched bars show the ratios of relative τmean values measured for long and short pulses, respectively. G, analogous results as presented in F but obtained for 3 μm of zolpidem. In F and G, mean values were calculated from at least n = 5 cells. Note that at low [GABA] flurazepam enhances the deactivation–desensitization coupling and zolpidem does not. Insets above current traces depict the time course of applied agonist. *Significant difference with respect to the control values.

Mentions: It is known that prolonged exposure of GABAARs to free agonist may result in a favoured entry into the desensitized state and therefore the time of agonist application may affect the deactivation kinetics (Jones & Westbrook, 1995). Taking into account this prediction, we have checked how time of agonist application affected deactivation kinetics of currents evoked by application at different [GABA] and how the ensuing deactivation–desensitization coupling is affected by flurazepam and zolpidem. To assess the deactivation–desensitization coupling at a given GABA concentration, currents were elicited by a GABA pulse of sufficient duration to reach the peak or a plateau value (a so called ‘short’ pulse; pulse durations are described in the legend of Fig. 5A–C) and then by a pulse at least five times longer (‘long’ pulses, Fig. 5A–C). The averaged deactivation time constants were measured for responses evoked by ‘long’ (τmean(long)) and ‘short’ pulses (τmean(short)) and the ratio of these time constants is shown in Fig. 5D. Interestingly, up to 10 μm GABA, the prolongation of agonist pulse did not affect the deactivation kinetics (Fig. 5D). On the contrary, starting from a GABA concentration of 30 μm, the larger [GABA] was, the larger the impact of time duration of the agonist pulse on the deactivation time course was (Fig. 5D). At high [GABA], a prolongation of the agonist pulse resulted in a reduction (or even disappearance) of the fast deactivation component (Fig. 5C) giving rise to a substantial increase in τmean (Fig. 5D). Indeed, while following 1 ms pulse of 10 mm GABA, there was a predominant fast component of ∼2.6 ms (see section above and Fig. 5C); after a longer pulse (10–30 ms), such a fast component was absent (Fig. 5C, right panel). Interestingly, the rapid decay component in currents evoked by 1 ms (10 mm GABA) and during a long pulse of the same [GABA] had indistinguishable fast decay components (see inset on the expanded time scale in Fig. 5C) indicating that the apparent fast deactivation is due to a rapid desensitization process. Indeed, as presented in details in the next section, the value of the fast desensitization time constant was not significantly different (P > 0.05) from the fast deactivation component. The view that the fast deactivation component is due to rapid desensitization is further supported by the fact that the recovery in the paired-pulse experiments with a short gap (two 1 ms pulses of 10 mm GABA separated by a 5 ms gap) yielded a recovery at the limit of detection level (below 5%, data not shown). Moreover, as already mentioned, the time courses of current responses elicited by 10 mm GABA pulses of duration between 1 and 3 ms were indistinguishable (data not shown) which is consistent with the mechanism in which even a very short pulse (e.g. 1 ms) of saturating [GABA] is sufficient to induce a profound desensitization that determines the fast component of apparent deactivation. However, the prolongation of deactivation process (at high [GABA]) by increasing the pulse duration was not only due to reduction or disappearance of the rapid component. For half-saturating or saturating [GABA], the slow deactivation components were significantly longer than the ones following the short pulses (Fig. 5E). Thus, at high [GABA], rapid desensitization appears to play a dual role in shaping the deactivation time course: (i) it largely determines the fast component of apparent deactivation and (ii) sojourns into the desensitized states prolong the slow component of deactivation process (Jones & Westbrook. 1995).


GABA transient sets the susceptibility of mIPSCs to modulation by benzodiazepine receptor agonists in rat hippocampal neurons.

Mozrzymas JW, Wójtowicz T, Piast M, Lebida K, Wyrembek P, Mercik K - J. Physiol. (Lond.) (2007)

Flurazepam and zolpidem differentially affect the deactivation–desensitization coupling A, B and C, examples of current traces evoked by 1 μm, 30 μm and 10 mm GABA, respectively, applied for short (thin line) and long time (thick line). The following durations of agonist applications were used at different [GABA]: 1 μm, 2 s; 3 μm, 1 s; 10 μm, 50 ms; 30 μm, 10 ms; 100 μm, 5 ms; 10 mm, 1 ms and the respective long applications were five times longer (see text for explanation of how short and long application times were defined). Right panels in A, B and C show the same current traces but normalized to the current value at the end of GABA pulse. In C, the inset shows the rapid phases of current responses on an expanded time scale. Note that at high [GABA] the deactivation following brief GABA pulse is much faster than that after a long application (mainly due to a predominant rapid deactivation component in the former one). D, the ratio of τmean values measured for deactivation currents following long and brief GABA applications, respectively, versus GABA concentration. Note that pulse duration affects τmean only for GABA concentrations equal or above 30 μm at which rapid desensitization becomes prominent. E, ratio of time constants of slow deactivation components (τslow) measured following long and brief GABA applications, respectively, versus[GABA]. In D and E, mean values were calculated from at least n = 8 cells. F, relative τmean for deactivation currents in the presence of 3 μm of flurazepam (with respect to control conditions) for short pulses (black bars) and for long pulses (middle hatched bars). Cross-hatched bars show the ratios of relative τmean values measured for long and short pulses, respectively. G, analogous results as presented in F but obtained for 3 μm of zolpidem. In F and G, mean values were calculated from at least n = 5 cells. Note that at low [GABA] flurazepam enhances the deactivation–desensitization coupling and zolpidem does not. Insets above current traces depict the time course of applied agonist. *Significant difference with respect to the control values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2375456&req=5

fig05: Flurazepam and zolpidem differentially affect the deactivation–desensitization coupling A, B and C, examples of current traces evoked by 1 μm, 30 μm and 10 mm GABA, respectively, applied for short (thin line) and long time (thick line). The following durations of agonist applications were used at different [GABA]: 1 μm, 2 s; 3 μm, 1 s; 10 μm, 50 ms; 30 μm, 10 ms; 100 μm, 5 ms; 10 mm, 1 ms and the respective long applications were five times longer (see text for explanation of how short and long application times were defined). Right panels in A, B and C show the same current traces but normalized to the current value at the end of GABA pulse. In C, the inset shows the rapid phases of current responses on an expanded time scale. Note that at high [GABA] the deactivation following brief GABA pulse is much faster than that after a long application (mainly due to a predominant rapid deactivation component in the former one). D, the ratio of τmean values measured for deactivation currents following long and brief GABA applications, respectively, versus GABA concentration. Note that pulse duration affects τmean only for GABA concentrations equal or above 30 μm at which rapid desensitization becomes prominent. E, ratio of time constants of slow deactivation components (τslow) measured following long and brief GABA applications, respectively, versus[GABA]. In D and E, mean values were calculated from at least n = 8 cells. F, relative τmean for deactivation currents in the presence of 3 μm of flurazepam (with respect to control conditions) for short pulses (black bars) and for long pulses (middle hatched bars). Cross-hatched bars show the ratios of relative τmean values measured for long and short pulses, respectively. G, analogous results as presented in F but obtained for 3 μm of zolpidem. In F and G, mean values were calculated from at least n = 5 cells. Note that at low [GABA] flurazepam enhances the deactivation–desensitization coupling and zolpidem does not. Insets above current traces depict the time course of applied agonist. *Significant difference with respect to the control values.
Mentions: It is known that prolonged exposure of GABAARs to free agonist may result in a favoured entry into the desensitized state and therefore the time of agonist application may affect the deactivation kinetics (Jones & Westbrook, 1995). Taking into account this prediction, we have checked how time of agonist application affected deactivation kinetics of currents evoked by application at different [GABA] and how the ensuing deactivation–desensitization coupling is affected by flurazepam and zolpidem. To assess the deactivation–desensitization coupling at a given GABA concentration, currents were elicited by a GABA pulse of sufficient duration to reach the peak or a plateau value (a so called ‘short’ pulse; pulse durations are described in the legend of Fig. 5A–C) and then by a pulse at least five times longer (‘long’ pulses, Fig. 5A–C). The averaged deactivation time constants were measured for responses evoked by ‘long’ (τmean(long)) and ‘short’ pulses (τmean(short)) and the ratio of these time constants is shown in Fig. 5D. Interestingly, up to 10 μm GABA, the prolongation of agonist pulse did not affect the deactivation kinetics (Fig. 5D). On the contrary, starting from a GABA concentration of 30 μm, the larger [GABA] was, the larger the impact of time duration of the agonist pulse on the deactivation time course was (Fig. 5D). At high [GABA], a prolongation of the agonist pulse resulted in a reduction (or even disappearance) of the fast deactivation component (Fig. 5C) giving rise to a substantial increase in τmean (Fig. 5D). Indeed, while following 1 ms pulse of 10 mm GABA, there was a predominant fast component of ∼2.6 ms (see section above and Fig. 5C); after a longer pulse (10–30 ms), such a fast component was absent (Fig. 5C, right panel). Interestingly, the rapid decay component in currents evoked by 1 ms (10 mm GABA) and during a long pulse of the same [GABA] had indistinguishable fast decay components (see inset on the expanded time scale in Fig. 5C) indicating that the apparent fast deactivation is due to a rapid desensitization process. Indeed, as presented in details in the next section, the value of the fast desensitization time constant was not significantly different (P > 0.05) from the fast deactivation component. The view that the fast deactivation component is due to rapid desensitization is further supported by the fact that the recovery in the paired-pulse experiments with a short gap (two 1 ms pulses of 10 mm GABA separated by a 5 ms gap) yielded a recovery at the limit of detection level (below 5%, data not shown). Moreover, as already mentioned, the time courses of current responses elicited by 10 mm GABA pulses of duration between 1 and 3 ms were indistinguishable (data not shown) which is consistent with the mechanism in which even a very short pulse (e.g. 1 ms) of saturating [GABA] is sufficient to induce a profound desensitization that determines the fast component of apparent deactivation. However, the prolongation of deactivation process (at high [GABA]) by increasing the pulse duration was not only due to reduction or disappearance of the rapid component. For half-saturating or saturating [GABA], the slow deactivation components were significantly longer than the ones following the short pulses (Fig. 5E). Thus, at high [GABA], rapid desensitization appears to play a dual role in shaping the deactivation time course: (i) it largely determines the fast component of apparent deactivation and (ii) sojourns into the desensitized states prolong the slow component of deactivation process (Jones & Westbrook. 1995).

Bottom Line: 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.We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroscience, Department of Biophysics, Wrocław Medical University, Chałubiñskiego 3, 50-368 Wroclaw, Poland.

ABSTRACT
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 GABA(A)R 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 GABA(A) 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') alpha1beta2gamma2 receptors reproduced all major findings observed for neuronal GABA(A)Rs. We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.

Show MeSH
Related in: MedlinePlus