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Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity.

Savtchenko L, Megalogeni M, Rusakov DA, Walker MC, Pavlov I - Nat Commun (2015)

Bottom Line: Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions.Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal.We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Neurology, Queen Square, London WC1N3BG, UK.

ABSTRACT
GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg(2+) model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity.

No MeSH data available.


Related in: MedlinePlus

GAT-1 does not reverse during epileptiform activity.(a) Changes in tonic GABAAR-mediated currents in CA1 pyramidal neurons following application of SKF899976A during ongoing epileptiform activity (n=6; PTX, picrotoxin; TTX, tetrodotoxin; error bars, s.e.m.). (b) The effect of GAT-1 inhibition on tonic GABAAR-mediated currents in CA1 pyramidal neurons in the absence of synaptic GABA release (slices preincubated in 1 μM concanamycin; n=6; error bars, s.e.m.). (c,d) Mean normalized traces of GABAAR transients (grey: s.e.m.) show that SKF89976A similarly prolongs burst-associated GABAAR transients in pyramidal neurons from control (c; area under the curve, AUC, increases from 88.8±18.2 to 188.6±53.9 ms; n=6; P=0.038, paired t-test) and epileptic hippocampi (d; AUC increase from 107.9±21.6 to 173.3±24.8 ms; n=8; P=0.0012, paired t-test). Bars, mean; error bars, s.e.m.; circles, individual experiments.
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f4: GAT-1 does not reverse during epileptiform activity.(a) Changes in tonic GABAAR-mediated currents in CA1 pyramidal neurons following application of SKF899976A during ongoing epileptiform activity (n=6; PTX, picrotoxin; TTX, tetrodotoxin; error bars, s.e.m.). (b) The effect of GAT-1 inhibition on tonic GABAAR-mediated currents in CA1 pyramidal neurons in the absence of synaptic GABA release (slices preincubated in 1 μM concanamycin; n=6; error bars, s.e.m.). (c,d) Mean normalized traces of GABAAR transients (grey: s.e.m.) show that SKF89976A similarly prolongs burst-associated GABAAR transients in pyramidal neurons from control (c; area under the curve, AUC, increases from 88.8±18.2 to 188.6±53.9 ms; n=6; P=0.038, paired t-test) and epileptic hippocampi (d; AUC increase from 107.9±21.6 to 173.3±24.8 ms; n=8; P=0.0012, paired t-test). Bars, mean; error bars, s.e.m.; circles, individual experiments.

Mentions: Does the tonic current in ‘epileptic’ slices result from increased synaptic GABA release or from the reversal of GABA transport? In the latter case, inhibiting GATs should decrease the extrasynaptic GABA concentrations and thus reduce the tonic conductance in hippocampal neurons. In contrast, application of SKF89976A resulted in an outward shift in Ihold (Fig. 4a; 27.9±7.1 pA, n=6, P=0.011, paired t-test) indicating that inhibition of GAT-1 increases GABAAR-mediated conductance in hippocampal neurons under conditions that promote the occurrence of epileptiform discharges. The effect of SKF89976A on the Ihold is qualitatively similar to that in the normal Mg2+ aCSF (that is, in the absence of epileptiform activity; 15.7±3.4 pA, n=5, P=0.0102, paired t-test; Supplementary Fig. 3). This increase is likely to be due to the accumulation of synaptically released GABA. To test this, we preincubated slices in 1 μM concanamycin and performed recordings in the presence of tetrodotoxin, that is, in conditions with no vesicular GABA release. Indeed, GAT-1 inhibition in these experiments had little effect on the Ihold, reflecting the lack of detectable GABA uptake in the absence of synaptic neurotransmission (Fig. 4b).


Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity.

Savtchenko L, Megalogeni M, Rusakov DA, Walker MC, Pavlov I - Nat Commun (2015)

GAT-1 does not reverse during epileptiform activity.(a) Changes in tonic GABAAR-mediated currents in CA1 pyramidal neurons following application of SKF899976A during ongoing epileptiform activity (n=6; PTX, picrotoxin; TTX, tetrodotoxin; error bars, s.e.m.). (b) The effect of GAT-1 inhibition on tonic GABAAR-mediated currents in CA1 pyramidal neurons in the absence of synaptic GABA release (slices preincubated in 1 μM concanamycin; n=6; error bars, s.e.m.). (c,d) Mean normalized traces of GABAAR transients (grey: s.e.m.) show that SKF89976A similarly prolongs burst-associated GABAAR transients in pyramidal neurons from control (c; area under the curve, AUC, increases from 88.8±18.2 to 188.6±53.9 ms; n=6; P=0.038, paired t-test) and epileptic hippocampi (d; AUC increase from 107.9±21.6 to 173.3±24.8 ms; n=8; P=0.0012, paired t-test). Bars, mean; error bars, s.e.m.; circles, individual experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374149&req=5

f4: GAT-1 does not reverse during epileptiform activity.(a) Changes in tonic GABAAR-mediated currents in CA1 pyramidal neurons following application of SKF899976A during ongoing epileptiform activity (n=6; PTX, picrotoxin; TTX, tetrodotoxin; error bars, s.e.m.). (b) The effect of GAT-1 inhibition on tonic GABAAR-mediated currents in CA1 pyramidal neurons in the absence of synaptic GABA release (slices preincubated in 1 μM concanamycin; n=6; error bars, s.e.m.). (c,d) Mean normalized traces of GABAAR transients (grey: s.e.m.) show that SKF89976A similarly prolongs burst-associated GABAAR transients in pyramidal neurons from control (c; area under the curve, AUC, increases from 88.8±18.2 to 188.6±53.9 ms; n=6; P=0.038, paired t-test) and epileptic hippocampi (d; AUC increase from 107.9±21.6 to 173.3±24.8 ms; n=8; P=0.0012, paired t-test). Bars, mean; error bars, s.e.m.; circles, individual experiments.
Mentions: Does the tonic current in ‘epileptic’ slices result from increased synaptic GABA release or from the reversal of GABA transport? In the latter case, inhibiting GATs should decrease the extrasynaptic GABA concentrations and thus reduce the tonic conductance in hippocampal neurons. In contrast, application of SKF89976A resulted in an outward shift in Ihold (Fig. 4a; 27.9±7.1 pA, n=6, P=0.011, paired t-test) indicating that inhibition of GAT-1 increases GABAAR-mediated conductance in hippocampal neurons under conditions that promote the occurrence of epileptiform discharges. The effect of SKF89976A on the Ihold is qualitatively similar to that in the normal Mg2+ aCSF (that is, in the absence of epileptiform activity; 15.7±3.4 pA, n=5, P=0.0102, paired t-test; Supplementary Fig. 3). This increase is likely to be due to the accumulation of synaptically released GABA. To test this, we preincubated slices in 1 μM concanamycin and performed recordings in the presence of tetrodotoxin, that is, in conditions with no vesicular GABA release. Indeed, GAT-1 inhibition in these experiments had little effect on the Ihold, reflecting the lack of detectable GABA uptake in the absence of synaptic neurotransmission (Fig. 4b).

Bottom Line: Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions.Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal.We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Neurology, Queen Square, London WC1N3BG, UK.

ABSTRACT
GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg(2+) model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity.

No MeSH data available.


Related in: MedlinePlus