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.

Keywords: GABA; ambient; extrasynaptic; inhibition; spillover.

FIGURE 1

Whole-cell sniffer with GABA_A receptors. (A) RT-PCR verifying the expression of GABA_A receptors composed of α1β2γ2 subunits in HEK cells. Primers for α6 and δ subunits were included as control. Expression of hGAPDH served as control (C) of cDNA quality. (B) Whole-cell recording of a HEK cell detached from the coverslip. (C) A single puff of GABA evoked a large outward current. (D1) Distribution of the amplitude of the evoked currents. 82% (14/17) of the HEK cells tested with GABA responded with large outward currents. (D2) Amplitude of the current evoked by repetitive GABA puffs applied every 60 s. The response was stable over hours (n = 2). (E) Response of two sniffers to GABA. The evoked current was suppressed by gabazine (10 μM) or by further addition of picrotoxin (50 μM).

FIGURE 2

Electrophysiological properties of the whole-cell sniffer. (A) I/V plot of sniffer responses to GABA (1 mM) from 7 cells clamped from -120 to 80 mV. Values were corrected for liquid junction potential. The average reversal potential (E_GABA) was of -67 mV. (B) Response of a sniffer to a prolonged exposure of GABA. The sniffer exhibited a non-inactivating component (double arrow). (C1) Responses of a sniffer to 1 s puff of increasing GABA concentrations (from 0.01 μM to 100 mM). (C2) Hill plot of dose–responses relationships of peak currents (r = 0.99994). Responses were normalized to the fitted I_max value. The average EC₅₀ for the peak response was 26.3 ± 3.0 μM (mean ± SE; n = 3).

FIGURE 3

Detection and quantification of ambient GABA in a slice preparation. (A1) Response of a sniffer at different positions above a slice preparation from the spinal cord. Positioning the sniffer at the slice surface evoked a persistent outward current and increased the noise of the recording. Both current and noise disappeared when the sniffer was moved 500 μm above the slice. The gray and black horizontal bars indicate the time intervals used for Fast Fourier Transformation (FFT) analysis in A2. (A2) Power spectrum densities of the current measured by the sniffer far from the slice (black) and on the surface (gray). The frequencies between 1 and 1000 Hz were stronger when the sniffer was just above the surface of the slice. (B1) Response of a sniffer positioned in a slice (gray background) or outside the slice (white background). The responses far from the slice were evoked by applying incremental GABA concentrations in the extracellular medium. TTX (50 nM) was added in the end of the experiment. (B2) Hill plot of concentration–response relationship of steady state currents evoked by GABA in the extracellular medium. The two open circles represent the measurements from the surface of the slice before and after calibration. The average concentration of GABA obtained from the curve was of 0.237 μM. The open diamond represents the measurement obtained in TTX.

FIGURE 4

Detection of GABA transients in a slice preparation. (A) Illustration of the experimental setup. GABA release was evoked either by puffing glutamate or uncaging glutamate with a 375 nm laser. The lower picture illustrates a sniffer filled with the fluorophore Alexa 488 (white) placed inside a slice from the spinal cord. The pipette coming from the right was used to puff glutamate (glu). (B) Examples of transient events recorded in the sniffer. (B1) Spontaneous events. (B2) Events evoked by glutamate uncaging. (B3) Events evoked by glutamate puff. The responses were abolished after addition of the GABA_A receptor antagonist gabazine (20 μM). (C1) Scheme of the preparation. Slice from the spinal cord with a dorsal root filament stimulated with a suction electrode. Sniffer positioned in the slice. (C2) Response of the sniffer to single shocks applied on the dorsal root filament. Dorsal root stimulation evoked transient release of GABA. The responses were abolished after addition of TTX (50 nM). (D) Characteristics of the different types of events recorded by the sniffer (evoked by GABA, spontaneous, evoked by glutamate or dorsal root stimulation). From left to right: amplitude; rise time of the rising phase (from 20 to 80% of peak); rate of rise calculated as current from 20 to 80% of peak divided by rise time from 20 to 80% of the peak; time constant (τ) of the of the decay estimated by fitting a single exponential function.