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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

Synaptic GABA release during epileptiform activity.(a) Regular stereotypic epileptiform discharges (average frequency: 0.15±0.05 Hz; n=3) induced in Mg2+-free aCSF. Insert: sample field potential recording traces. (b) An example of a large GABAAR-mediated current in a CA1 pyramidal neuron (black, whole-cell voltage clamp, Vhold=0 mV) associated with a field potential (f.p.) burst (blue). (c) Typical recording of GABAAR-mediated drive onto a pyramidal neuron (Vhold=0 mV) during epileptiform activity. PTX, picrotoxin; TTX, tetrodotoxin. (d) Time course of Ihold changes induced by co-application of SKF89976A and SNAP5114 in control and Mg2+-free aCSF (rate of Ihold increase calculated 1–5 min after GAT inhibitors application in 0 Mg2+: 1.1±0.2 pA s−1; in control: 0.16±0.04 pA s−1; n=4 for each condition; P=0.004, t-test). (e) GAT inhibitors suppress the frequency of epileptiform discharges (3.6±2.6% of baseline; n=4, P<0.001, paired t-test). Error bars, s.e.m.
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f3: Synaptic GABA release during epileptiform activity.(a) Regular stereotypic epileptiform discharges (average frequency: 0.15±0.05 Hz; n=3) induced in Mg2+-free aCSF. Insert: sample field potential recording traces. (b) An example of a large GABAAR-mediated current in a CA1 pyramidal neuron (black, whole-cell voltage clamp, Vhold=0 mV) associated with a field potential (f.p.) burst (blue). (c) Typical recording of GABAAR-mediated drive onto a pyramidal neuron (Vhold=0 mV) during epileptiform activity. PTX, picrotoxin; TTX, tetrodotoxin. (d) Time course of Ihold changes induced by co-application of SKF89976A and SNAP5114 in control and Mg2+-free aCSF (rate of Ihold increase calculated 1–5 min after GAT inhibitors application in 0 Mg2+: 1.1±0.2 pA s−1; in control: 0.16±0.04 pA s−1; n=4 for each condition; P=0.004, t-test). (e) GAT inhibitors suppress the frequency of epileptiform discharges (3.6±2.6% of baseline; n=4, P<0.001, paired t-test). Error bars, s.e.m.

Mentions: To address whether GABA transport reverses during extremes of network activity, we induced regular recurrent epileptiform discharges by using Mg2+-free artificial cerebrospinal fluid (aCSF)35. Under these conditions, the majority of slices developed stereotypical spontaneous interictal-like field potential bursts that persisted at a stable frequency for at least 1 h (Fig. 3a). To monitor the GABAAR-mediated currents in neurons during on-going epileptiform activity, pyramidal cells were voltage clamped at 0 mV, close to the reversal potential of glutamate receptor-mediated currents, using an intracellular solution containing 8 mM Cl− (calculated Cl− reversal potential, ECl=−72 mV). Simultaneous field potential and whole-cell recordings revealed that each field potential burst was accompanied by a concomitant GABAAR-mediated transient (Fig. 3b,c). These GABAAR-mediated currents in pyramidal cells reached up to 1.5–2 nA in amplitude, while, between bursts, spontaneously occurring inhibitory post-synaptic currents (IPSCs) were two orders of magnitude smaller. We also noted that field potential recordings underestimate the duration of underlying burst discharges as these large, interictal-like event-associated GABAergic currents were considerably longer than bursts detected by extracellular electrodes, and often burst-associated large IPSCs were heralded by a flurry of smaller amplitude IPSCs at high frequencies (Fig. 3c).


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)

Synaptic GABA release during epileptiform activity.(a) Regular stereotypic epileptiform discharges (average frequency: 0.15±0.05 Hz; n=3) induced in Mg2+-free aCSF. Insert: sample field potential recording traces. (b) An example of a large GABAAR-mediated current in a CA1 pyramidal neuron (black, whole-cell voltage clamp, Vhold=0 mV) associated with a field potential (f.p.) burst (blue). (c) Typical recording of GABAAR-mediated drive onto a pyramidal neuron (Vhold=0 mV) during epileptiform activity. PTX, picrotoxin; TTX, tetrodotoxin. (d) Time course of Ihold changes induced by co-application of SKF89976A and SNAP5114 in control and Mg2+-free aCSF (rate of Ihold increase calculated 1–5 min after GAT inhibitors application in 0 Mg2+: 1.1±0.2 pA s−1; in control: 0.16±0.04 pA s−1; n=4 for each condition; P=0.004, t-test). (e) GAT inhibitors suppress the frequency of epileptiform discharges (3.6±2.6% of baseline; n=4, P<0.001, paired t-test). Error bars, s.e.m.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374149&req=5

f3: Synaptic GABA release during epileptiform activity.(a) Regular stereotypic epileptiform discharges (average frequency: 0.15±0.05 Hz; n=3) induced in Mg2+-free aCSF. Insert: sample field potential recording traces. (b) An example of a large GABAAR-mediated current in a CA1 pyramidal neuron (black, whole-cell voltage clamp, Vhold=0 mV) associated with a field potential (f.p.) burst (blue). (c) Typical recording of GABAAR-mediated drive onto a pyramidal neuron (Vhold=0 mV) during epileptiform activity. PTX, picrotoxin; TTX, tetrodotoxin. (d) Time course of Ihold changes induced by co-application of SKF89976A and SNAP5114 in control and Mg2+-free aCSF (rate of Ihold increase calculated 1–5 min after GAT inhibitors application in 0 Mg2+: 1.1±0.2 pA s−1; in control: 0.16±0.04 pA s−1; n=4 for each condition; P=0.004, t-test). (e) GAT inhibitors suppress the frequency of epileptiform discharges (3.6±2.6% of baseline; n=4, P<0.001, paired t-test). Error bars, s.e.m.
Mentions: To address whether GABA transport reverses during extremes of network activity, we induced regular recurrent epileptiform discharges by using Mg2+-free artificial cerebrospinal fluid (aCSF)35. Under these conditions, the majority of slices developed stereotypical spontaneous interictal-like field potential bursts that persisted at a stable frequency for at least 1 h (Fig. 3a). To monitor the GABAAR-mediated currents in neurons during on-going epileptiform activity, pyramidal cells were voltage clamped at 0 mV, close to the reversal potential of glutamate receptor-mediated currents, using an intracellular solution containing 8 mM Cl− (calculated Cl− reversal potential, ECl=−72 mV). Simultaneous field potential and whole-cell recordings revealed that each field potential burst was accompanied by a concomitant GABAAR-mediated transient (Fig. 3b,c). These GABAAR-mediated currents in pyramidal cells reached up to 1.5–2 nA in amplitude, while, between bursts, spontaneously occurring inhibitory post-synaptic currents (IPSCs) were two orders of magnitude smaller. We also noted that field potential recordings underestimate the duration of underlying burst discharges as these large, interictal-like event-associated GABAergic currents were considerably longer than bursts detected by extracellular electrodes, and often burst-associated large IPSCs were heralded by a flurry of smaller amplitude IPSCs at high frequencies (Fig. 3c).

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