Lack of an endogenous GABAA receptor-mediated tonic current in hypoglossal motoneurons

Abstract

Tonic GABAA receptor-mediated current is an important modulator of neuronal excitability, but it is not known if it is present in mammalian motoneurons. To address this question studies were performed using whole-cell patch-clamp recordings from mouse hypoglossal motoneurons (HMs) in an in vitro slice preparation. In the presence of blockers of glutamatergic and glycinergic receptor-mediated transmission application of SR-95531 or bicuculline, while abolishing GABAA receptor-mediated phasic synaptic currents, did not reveal a tonic GABAA receptor-mediated current. Additionally, blockade of both GAT-1 and GAT-3 GABA transporters did not unmask this tonic current. In contrast, application of exogenous GABA (1 to 15 μm) resulted in a tonic GABAergic current that was observed when both GAT-1 and GAT-3 transporters were simultaneously blocked, and this current was greater than the sum of the current observed when each transporter was blocked individually. We also investigated which GABAA receptor subunits may be responsible for the current. Application of the δ subunit GABAA receptor agonist THIP resulted in a tonic GABAA receptor current. Application of the δ subunit modulator THDOC resulted in an enhanced tonic current. Application of the α5 subunit GABAA receptor inverse agonist L-655,708 did not modulate the current. In conclusion, these data show that HMs have tonic GABAA receptor-mediated current. The level of GABA in the vicinity of GABAA receptors responsible for this current is regulated by GABA transporters. In HMs a tonic current in response to exogenous GABA probably arises from activation of GABAA receptors containing δ subunits.

Figure 1. Application of the GAT-1 GABA transporter blocker SKF 89976A (30 μm) and the GAT-3 blocker SNAP 5114 (50 μm) did not unmask an endogenous BMI-sensitive tonic GABA_A receptor-mediated current in a HM

To the left of the current trace are two all-points histograms each with a normal distribution fitted to the region of the smoothed histogram that was not contaminated by large synaptic events. Also shown by the dashed lines are the means of each normal distribution (I_Hold). Blue coloured histogram and dashed line are for the control condition (before application of BMI, mean I_Hold=−128.9 ± 2.9 pA) and the red coloured histogram and dashed line are after application of BMI (I_Hold=−128.6 ± 2.7 pA). Note that the BMI (20 μm) did abolish the phasic spontaneous inhibitory postsynaptic currents. Data from a HM voltage clamped at –70 mV in a P5 mouse (with symmetrical Cl⁻ concentration inside and outside the pipette); also the slice was continuously bathed with an ACSF containing blockers of glutamate and glycine ligand-gated receptors (basic blockers).

Figure 2. The unmasking of a BMI-sensitive GABA_A receptor-mediated tonic current in the presence of exogenous GABA (5 μm) is dependent on blockade of GABA transporters

A, application of exogenous GABA in the presence of the GAT-1 GABA transporter blocker SKF 89976A (30 μm) does not evoke a BMI-sensitive tonic GABA_A receptor-mediated current in a HM. Data from a P10 mouse. B, application of exogenous GABA in the presence of the GAT-3 GABA transporter blocker SNAP 5114 (50 μm) evokes a BMI-sensitive tonic GABA_A receptor-mediated current in a HM. Data from a P5 mouse. C, application of exogenous GABA in the presence of both GAT-1 and GAT-3 transporter blockers results in a robust BMI-sensitive tonic GABA_A receptor-mediated current. Data from a P9 mouse. In A, B and C HMs voltage-clamped at –70 mV, and the slices were continuously bathed with an ACSF solution containing TTX, blockers of glutamate and glycine ligand-gated receptors, and a blocker of GABA_B receptors. As in Fig. 1, to the left of each current trace are two all-points histograms each with a normal distribution fitted to the smoothed histogram. Also shown by the dashed lines are the means of each normal distribution (I_Hold). Blue coloured histogram and dashed line are for the control condition (before application of BMI) and the red coloured histogram and dashed line are for after application of BMI. D, summary data for the change in holding current (in the presence of 5 μm GABA) in BMI compared with control with blockade of GAT-1 alone, GAT-3 alone, and combined blockade of GAT-1 and GAT-3. Denoted is whether ΔI_Hold was significantly different from zero: ns indicates no change; *significance at a P < 0.05 level; **significance at a P < 0.005 level. Number below each treatment indicates the sample size. Shown are the mean values for each treatment while the error bars indicate SEM.

Figure 3. GABA dose–response data for the tonic GABA-mediated current

In this case the GABA-activated tonic current was measured as the difference between the mean membrane current before application of GABA and the mean current measured during GABA application (see text for justification). Data shown are means ± SEM, and the number next to each data point indicates the number of HMs studied at each GABA concentration. All HMs voltage-clamped at –70 mV, and the slices were continuously bathed with an ACSF containing TTX, blockers of glutamate and glycine ligand-gated receptors, a blocker of GABA_B receptors and both GAT-1 and GAT-3 transporter blockers.

Figure 4. GABA_A receptor subunits responsible for the tonic GABA current

A, application of the α₅-subunit-sensitive inverse agonist L-655,708 (20 μm) did not decrease the BMI-sensitive tonic current seen with application of exogenous GABA in the presence of the GAT-1 and GAT-3 GABA transporter blockers. Data from a P5 mouse, with the HM voltage-clamped at –70 mV, and the slice was continuously bathed with an ACSF containing TTX, blockers of glutamate and glycine ligand-gated receptors, and a blocker of GABA_B receptors. To the right of the current trace are three all-points histograms, each with a normal distribution fitted to the smoothed histogram. Also shown by the dashed lines are the means of each normal distribution (I_Hold). Blue coloured histogram and dashed line are for GABA, green coloured histogram and dashed line are for GABA + L-655,708 (immediately before application of BMI) and the red coloured histogram and dashed line are for after application of BMI. B, the GABA_A receptor neurosteroid modulator THDOC (500 nm) enhances the BMI-sensitive GABA_A receptor-mediated tonic current. Application of the δ-subunit-sensitive neurosteroid modulator THDOC increased the tonic current seen with application of exogenous GABA (2 μm) in the presence of the GAT-1 and GAT-3 GABA transporter blockers. Data from a P6 mouse, with the HM voltage-clamped at –70 mV, and the slice was continuously bathed with an ACSF containing TTX, blockers of glutamate and glycine ligand-gated receptors, and a blocker of GABA_B receptors. To the right of the current trace are three all-points histograms each with a normal distribution fitted to the smoothed histogram. Also shown by the dashed lines are the means of each normal distribution (I_Hold). Blue coloured histogram and dashed line are in GABA, green coloured histogram and dashed line are in GABA + THDOC (immediately before application of BMI) and the red coloured histogram and dashed line are after application of BMI. C, the δ-subunit GABA_A receptor agonist THIP activates a BMI-sensitive GABA_A receptor-mediated tonic current. Application of the δ-subunit agonist THIP (4 μm) resulted in a BMI-sensitive tonic current. Data from a P6 mouse, with the HM voltage-clamped at –70 mV, and the slice was continuously bathed with an ACSF containing blockers of glutamate and glycine ligand-gated receptors. To the right of the current trace are two all-points histograms each with a normal distribution fitted to the smoothed histogram. Also shown by the dashed lines are the means of each normal distribution (I_Hold). Blue coloured histogram and dashed line are in THIP (immediately before application of BMI) and the red coloured histogram and dashed line are after application of BMI.