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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

Tonic GABAAR currents in the presence of GAT inhibitors.(a) Top: a typical trace showing the effect of GAT-3 inhibitor SNAP5114 (100 μM) on the holding current (Ihold) of a hippocampal CA1 pyramidal neuron. Bottom: averaged time course of five experiments. Data points in red indicate drug application times; PTX, picrotoxin. (b) Top: a typical experiment in which tonic GABAAR-mediated currents were induced by consequent application of GAT-1 (SKF89976A; 30 μM) and GAT-3 (SNAP5114; 100 μM) inhibitors. Bottom: averaged time course of six experiments. Data points in red indicate drug application times. (c) Box plot (boxes, 25–75%; whiskers, minimum–maximum, lines, median; × , mean) showing changes in Ihold caused by application of GAT inhibitors (SNAP5114: n=5, P=0.3, compared with baseline, paired t-test; SKF89976A: n=11, P=0.0002, compared with baseline, paired t-test; SKF89976A+SNAP5114: n=6, P=0.0014, compared with SKF89976A alone, paired t-test). Note high [Cl−]in internal solution in these experiments (Vhold=−70 mV); NS, not significant; *, significant changes; error bars on time course plots, s.e.m.
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f2: Tonic GABAAR currents in the presence of GAT inhibitors.(a) Top: a typical trace showing the effect of GAT-3 inhibitor SNAP5114 (100 μM) on the holding current (Ihold) of a hippocampal CA1 pyramidal neuron. Bottom: averaged time course of five experiments. Data points in red indicate drug application times; PTX, picrotoxin. (b) Top: a typical experiment in which tonic GABAAR-mediated currents were induced by consequent application of GAT-1 (SKF89976A; 30 μM) and GAT-3 (SNAP5114; 100 μM) inhibitors. Bottom: averaged time course of six experiments. Data points in red indicate drug application times. (c) Box plot (boxes, 25–75%; whiskers, minimum–maximum, lines, median; × , mean) showing changes in Ihold caused by application of GAT inhibitors (SNAP5114: n=5, P=0.3, compared with baseline, paired t-test; SKF89976A: n=11, P=0.0002, compared with baseline, paired t-test; SKF89976A+SNAP5114: n=6, P=0.0014, compared with SKF89976A alone, paired t-test). Note high [Cl−]in internal solution in these experiments (Vhold=−70 mV); NS, not significant; *, significant changes; error bars on time course plots, s.e.m.

Mentions: The relative contribution of the main cortical GABA transporters GAT-1 (neuronal) and GAT-3 (glial) to GABA uptake varies depending on brain region and cell type5173334. We first tested their impact on [GABA]e in the hippocampal area CA1 using GAT inhibitors (Fig. 2). Whole-cell patch-clamp recordings were performed from pyramidal neurons in the presence of CGP55845 (1 μM), NBQX (20 μM), APV (50 μM) and tetrodotoxin (1 μM) to block GABAB and ionotropic glutamate receptors, as well as action potential-dependent GABA release. Neurons were held at −70 mV and recorded using high Cl− internal solution (see Methods). Application of the GAT-1 inhibitor SKF89976A (30 μM) produced a significant (Holm–Bonferroni correction) inward shift of holding current (ΔIhold; −27.7±4.7 pA; n=11, P=0.0002, paired t-test; Fig. 2b,c). No apparent significant (Holm–Bonferroni correction) changes in Ihold were observed on application of the GAT-3 inhibitor SNAP5114 (100 μM; −2.2±1.9 pA; n=5, P=0.3, paired t-test; Fig. 2a,c), confirming that under baseline conditions, with GAT-1 operational, GAT-3 has little effect on the GABA concentration detected by principal neurons in the hippocampus1719. The same concentration of SNAP5114 applied following inhibition of the GAT-1 transporter resulted in a rapid significant (Holm–Bonferroni correction) increase in the inward current, indicating that GAT-3-mediated GABA uptake regulates [GABA]e around pyramidal cells in the absence of GAT-1 activity (ΔIhold: −113.7±17.8 pA; n=6, P=0.0014, paired t-test compared with SKF89976A alone; Fig. 2b,c).


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)

Tonic GABAAR currents in the presence of GAT inhibitors.(a) Top: a typical trace showing the effect of GAT-3 inhibitor SNAP5114 (100 μM) on the holding current (Ihold) of a hippocampal CA1 pyramidal neuron. Bottom: averaged time course of five experiments. Data points in red indicate drug application times; PTX, picrotoxin. (b) Top: a typical experiment in which tonic GABAAR-mediated currents were induced by consequent application of GAT-1 (SKF89976A; 30 μM) and GAT-3 (SNAP5114; 100 μM) inhibitors. Bottom: averaged time course of six experiments. Data points in red indicate drug application times. (c) Box plot (boxes, 25–75%; whiskers, minimum–maximum, lines, median; × , mean) showing changes in Ihold caused by application of GAT inhibitors (SNAP5114: n=5, P=0.3, compared with baseline, paired t-test; SKF89976A: n=11, P=0.0002, compared with baseline, paired t-test; SKF89976A+SNAP5114: n=6, P=0.0014, compared with SKF89976A alone, paired t-test). Note high [Cl−]in internal solution in these experiments (Vhold=−70 mV); NS, not significant; *, significant changes; error bars on time course plots, s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374149&req=5

f2: Tonic GABAAR currents in the presence of GAT inhibitors.(a) Top: a typical trace showing the effect of GAT-3 inhibitor SNAP5114 (100 μM) on the holding current (Ihold) of a hippocampal CA1 pyramidal neuron. Bottom: averaged time course of five experiments. Data points in red indicate drug application times; PTX, picrotoxin. (b) Top: a typical experiment in which tonic GABAAR-mediated currents were induced by consequent application of GAT-1 (SKF89976A; 30 μM) and GAT-3 (SNAP5114; 100 μM) inhibitors. Bottom: averaged time course of six experiments. Data points in red indicate drug application times. (c) Box plot (boxes, 25–75%; whiskers, minimum–maximum, lines, median; × , mean) showing changes in Ihold caused by application of GAT inhibitors (SNAP5114: n=5, P=0.3, compared with baseline, paired t-test; SKF89976A: n=11, P=0.0002, compared with baseline, paired t-test; SKF89976A+SNAP5114: n=6, P=0.0014, compared with SKF89976A alone, paired t-test). Note high [Cl−]in internal solution in these experiments (Vhold=−70 mV); NS, not significant; *, significant changes; error bars on time course plots, s.e.m.
Mentions: The relative contribution of the main cortical GABA transporters GAT-1 (neuronal) and GAT-3 (glial) to GABA uptake varies depending on brain region and cell type5173334. We first tested their impact on [GABA]e in the hippocampal area CA1 using GAT inhibitors (Fig. 2). Whole-cell patch-clamp recordings were performed from pyramidal neurons in the presence of CGP55845 (1 μM), NBQX (20 μM), APV (50 μM) and tetrodotoxin (1 μM) to block GABAB and ionotropic glutamate receptors, as well as action potential-dependent GABA release. Neurons were held at −70 mV and recorded using high Cl− internal solution (see Methods). Application of the GAT-1 inhibitor SKF89976A (30 μM) produced a significant (Holm–Bonferroni correction) inward shift of holding current (ΔIhold; −27.7±4.7 pA; n=11, P=0.0002, paired t-test; Fig. 2b,c). No apparent significant (Holm–Bonferroni correction) changes in Ihold were observed on application of the GAT-3 inhibitor SNAP5114 (100 μM; −2.2±1.9 pA; n=5, P=0.3, paired t-test; Fig. 2a,c), confirming that under baseline conditions, with GAT-1 operational, GAT-3 has little effect on the GABA concentration detected by principal neurons in the hippocampus1719. The same concentration of SNAP5114 applied following inhibition of the GAT-1 transporter resulted in a rapid significant (Holm–Bonferroni correction) increase in the inward current, indicating that GAT-3-mediated GABA uptake regulates [GABA]e around pyramidal cells in the absence of GAT-1 activity (ΔIhold: −113.7±17.8 pA; n=6, P=0.0014, paired t-test compared with SKF89976A alone; Fig. 2b,c).

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