Fast detection of extrasynaptic GABA with a whole-cell sniffer

Abstract

Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a “sniffer” allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations.

GABA is a modulator, rather than a classical transmitter, in the medial nucleus of the trapezoid body–lateral superior olive sound localization circuit

“…Because of the activity dependency, measuring ambient GABA is challenging, and the diversity of experimental conditions and analytical methods makes comparisons of tonic inhibition between studies quite difficult (Bright & Smart, ; Christensen et al . ).…”

“…The assumption is consistent with previous reports demonstrating a dependency of ambient GABA levels on neuronal activity (Lerma et al 1986;Rowley et al 1995;Kennedy et al 2002;de Groote & Linthorst, 2007;Vanini et al 2011). Because of the activity dependency, measuring ambient GABA is challenging, and the diversity of experimental conditions and analytical methods makes comparisons of tonic inhibition between studies quite difficult (Bright & Smart, 2013;Christensen et al 2014).…”

Linking Metabolism to Membrane Signaling: The GABA–Malate Connection

“…A GABA sensor that allows time-resolved micromolar spatial resolution in living tissue would be a great benefit to explore how GABA compartmentation is regulated. Some progress has occurred using whole-cell patch clamp with human embryonic kidney cells expressing GABA receptors [61] or GABA imprinted electrodes [62].…”

Spinal dorsal horn astrocytes release GABA in response to synaptic activation

“…By varying the concentration of GABA in the puff pipette, we found that the responses increased monotonically with GABA and saturated at concentrations above 1 m m (Christensen et al . ). These results demonstrate that our sensor can be used to detect GABA transients.…”

“…HEK cells recorded in voltage-clamp mode transduced extracellular GABA into an electrical current (Christensen et al 2014). When held at 0 mV, the GABA sniffer responded to brief GABA puff (1 mM) by strong transient outward currents that were blocked by gabazine (10 μM) and picrotoxin (50 μM) ( Fig.…”