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The Journal of Physiology logoLink to The Journal of Physiology
. 2012 Apr 10;590(Pt 13):2965–2976. doi: 10.1113/jphysiol.2012.231944

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

J M Numata 1, J F M van Brederode 1, A J Berger 1
PMCID: PMC3406384  PMID: 22495589

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.

Key points

  • Tonic GABAA receptor-mediated currents have profound effects on neuronal excitability, yet it is not known whether this current is present in mammalian motoneurons.

  • This study shows that tonic GABAA receptor-mediated current can be observed in hypoglossal motoneurons in vitro under certain experimental conditions.

  • The tonic current was only observed when exogenous GABA was applied and GABA transporters were blocked suggesting that GABA transporters highly regulate extracellular GABA concentration.

  • Furthermore, we demonstrate that the current probably arises from activation of extrasynaptic GABAA receptors containing a δ subunit.

  • This tonic current may function to reduce the excitability of hypoglossal motoneurons; these motoneurons are important in many functions including chewing, swallowing, suckling, vocalization and upper airway patency.

Introduction

The ligand-gated GABAA receptor is a pentameric assembly of subunits drawn from almost 20 identified subunits (Farrant & Nusser, 2005; Farrant & Kaila, 2007). In brain the subunit assemblies that mediate phasic synaptic inhibition are primarily composed of α1β2/3γ2, α2β2/3γ2 or α3β2/3γ2, whereas GABAA receptors that mediate so-called GABA tonic inhibition are composed of α4βxδ, α6βxδ or α5βxγ2. GABAA receptors containing the δ subunit are found exclusively in extrasynaptic and perisynaptic locations (Farrant & Kaila, 2007). Generally, but not exclusively, the assemblies that mediate phasic synaptic inhibition have higher GABA EC50 values than those that mediate tonic inhibition (Farrant & Nusser, 2005; Farrant & Kaila, 2007; Glykys & Mody, 2007). Thus, activation of extrasynaptic GABAA receptors that mediate tonic inhibition occurs at lower GABA concentrations compared with GABA concentrations that activate phasic synaptic GABAA receptors.

The concentration of GABA in the extracellular space is governed by a number of factors including GABA transporters. It has been reported that tonic GABA currents are sensitive to GABA uptake by transporters (Semyanov et al. 2003; Glykys & Mody, 2007). Recently it has been shown both in neocortex and in the dorsal motor nucleus of the vagus that the GAT-1 and GAT-2/3 transporters both act to regulate extracellular GABA concentration (Keros & Hablitz, 2005; Gao & Smith, 2010).

While glycine is the predominant inhibitory neurotransmitter in spinal cord and brainstem, GABA is also present. Phasic GABAergic synaptic transmission to motoneurons is readily observed (Gao et al. 1998; Jonas et al. 1998; O’Brien & Berger, 1999, 2001; Donato & Nistri, 2000). A considerable amount of information is known about tonic GABAergic inhibition in a number of regions of the brain, including the cerebellum (Brickley et al. 1996; Stell et al. 2003), dentate gyrus (Stell & Mody, 2002; Zhan & Nadler, 2009), hippocampus (Semyanov et al. 2003; Caraiscos et al. 2004; Glykys & Mody, 2006) and thalamus (Cope et al. 2005; Bright et al. 2007), where GABA is the primary inhibitory neurotransmitter. Surprisingly, there is an absence of experimental data demonstrating the presence of GABA-mediated tonic inhibition of mammalian somatic motoneurons located in either the spinal cord or brainstem (Donato & Nistri, 2000). This is in contrast to glycine receptor-mediated tonic inhibition, where in glycine transporter subtype 1 (GlyT1)-deficient mice it has been shown that a strychnine-sensitive glycine receptor-mediated tonic current can be observed in hypoglossal motoneurons (HMs) (Gomeza et al. 2003).

Our previous work (Van Brederode et al. 2011) demonstrated that an important source of GABAergic input to HMs arises from GABAergic neurons that occupy a dense collection of cells just ventrolateral to the hypoglossal nucleus; this collection is called the Nucleus of Roller. We showed, in the in vitro brainstem slice that exhibits rhythmic respiratory activity, that many of these Nucleus of Roller GABAergic neurons are not phasically active but are tonically active (Van Brederode et al. 2011). Such tonic spike activity may provide an important source of synaptically released GABA in the hypoglossal motor nucleus. Therefore, we wondered whether HMs exhibit a tonic GABAA receptor-mediated current. In the present study we determined whether HMs recorded in vitro in brainstem slices show tonic GABAA receptor-mediated current and investigated which GABAA receptor subunits maybe involved in the generation of this current. A portion of these results have been presented in abstract form (Numata et al. 2010).

Methods

Ethical approval

Experiments were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC) and strictly followed the guidelines set out by the National Institutes of Health Guidelines for the Use and Care of Laboratory Animals.

Experimental procedures

In vitro experiments were performed on medullary slices derived from a total of 57 neonatal (P3 to P15) mouse pups. Mice were anaesthetized with isoflurane and killed by decapitation. The brainstem was quickly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (mm): 118 NaCl, 3 KCl, 1 NaH2PO4, 25 NaHCO3, 1.5 CaCl2, and 30 dextrose that was continuously gassed with a 95% O2 and 5% CO2 mixture (carbogen). Transverse slices (200–250 μm thick) were cut using a vibratome (Pelco). Slices were then incubated in ACSF for 1 h at 32°C prior to recording. Following incubation, slices were placed in a holding chamber that contained ACSF gassed with carbogen held at room temperature before being transferred to the heated recording chamber. Using an upright microscope (Zeiss, Axioskope) HMs in slices were visualized using infra-red differential interference contrast optics.

All whole-cell recordings of GABAergic currents were performed at a bath temperature of ∼30°C in the presence of blockers (termed basic blockers) of AMPA-mediated (blocked with DNQX, 10 μm, Sigma), NMDA-mediated (blocked with d-AP5, 50 μm, Tocris) and glycinergic (blocked with strychnine, 1 μm, Sigma) synaptic transmission. Additionally, when exogenous GABA was added to the bathing solution GABAB receptors were blocked with CGP 55845 (1 μm, Tocris), and action potentials were blocked with tetrodotoxin (TTX, 1 μm, Alomone Labs).

Patch recording electrodes were pulled from thin-walled borosilicate glass to a resistance of 2.5–4 MΩ. Electrodes were filled with a CsCl-based internal solution that contained either (in mm): 130 CsCl, 1 CaCl2, 3.45 Cs-BAPTA, 10 Hepes, 5 MgATP and 10 QX-314; or 140 CsCl, 1 MgCl2, 4 NaCl, 0.1 EGTA, 10 Hepes, 2 MgATP, 0.3 NaATP and 5 QX-314 (both solutions had an adjusted osmolarity of 290 mosmol l−1, and the pH was adjusted to 7.3). Voltage-clamp recordings were made with an Axopatch 200B amplifier (Molecular Devices). We visually identified HMs as large multipolar cells located in the hypoglossal motor nucleus as previously described in publications from this laboratory (Viana et al. 1993). The experimental protocol consisted of recording membrane current under voltage-clamp conditions from a HM held at −70 mV. Voltage-clamp recordings were not corrected for the small liquid junction potentials. At this holding potential GABAergic currents (both synaptic and tonic) are inward due to the high Cl− concentration of the CsCl-based internal solution. After establishment of whole-cell recording configuration, the carbogen-gassed ACSF was switched to one containing the basic blockers (see above). Following approximately 5 min in basic blockers, different reagents, depending on the experiment, were added to the ACSF containing basic blockers. Whole-cell currents were low-pass filtered at 5 kHz and digitally sampled at 20 kHz using pCLAMP10 (Molecular Devices). Data were acquired from only one HM in any single slice in order to avoid issues associated with incomplete washout of reagents or incomplete recovery back to baseline conditions.

Analysis of tonic current

Quantitative assessment of tonic GABA current was done using a method similar to that described by Glykys and Mody (2006). Based on their procedure we wrote a macro in IGOR Pro (Wavemetrics) to extract values of membrane current. Briefly, entire membrane current recordings were split into 20,000 point segments, each segment corresponding to 1 s of data. An all-points histogram was created for each 1 s segment. The histograms were smoothed with the Savitzky–Golay algorithm. A normal distribution was fitted to the region of the smoothed histogram that was not contaminated by large synaptic events; the latter were present in the long tail of the histogram. For each of the 1 s segments, we determined the mean of each normal distribution (IHold). For each treatment the average value of membrane current was based upon the average of the mean of the normal distributions for data taken from a 30 s period during the treatment. The determination of whether a GABAA receptor-mediated tonic current (ΔIHold) was present was based upon the computed value of the average membrane current after blocking GABAA receptors with either bicuculline methiodide (BMI, 20 μm, Sigma) or with gabazine (SR 95531, 20 μm, Tocris) compared with the current before application of these GABAA receptor blockers. Other drugs added in various experiments included: SKF 89976A (30 μm, Tocris), SNAP 5114 (50 μm, Tocris), THIP (4 μm, Tocris), THDOC (500 nm, Sigma) and L-655708 (20 μm, Sigma).

Statistics

Statistical comparisons were performed using the Student's two-tailed t test (paired comparisons unless otherwise noted) with significance set at P < 0.05. Results are expressed as mean ± SEM unless otherwise noted.

Results

In the absence of exogenous GABA a tonic GABA current is not observed

Initially we investigated whether in the in vitro slice preparation HMs exhibit an endogenous GABAA receptor-mediated tonic current. In the first series of experiments, we blocked all non-GABAergic ligand-gated fast synaptic transmission by applying to the ACSF bathing solution the basic blockers described above to block glutamatergic and glycinergic receptors. Under these conditions bath-applied SR 95531 (20 μm) did abolish GABAA receptor-mediated phasic inhibitory postsynaptic currents, but it did not result in a change in the holding current (ΔIHold) of the neurons. SR 95531-sensitive changes in the holding current are regarded as showing the presence of a tonic GABAA receptor-mediated current (Semyanov et al. 2004; Farrant & Nusser, 2005). This result of no significant change in IHold was seen in six HMs (ΔIHold=–9.2 ± 4.7 pA, P > 0.05, data not shown). This indicated that blockade of GABAA receptors alone with SR 95531 does not uncover an endogenously active tonic GABA current. This negative result could be explained by a number of factors. These include the use of a superfused slice preparation that can result in the lowering of the endogenous GABA concentration due to the washing away of GABA in the perfusate. Alternatively, but not exclusively, it could be due to the activity of GABA transporters resulting in a low concentration of extracellular GABA.

Next we pharmacologically blocked two of the GABA transporters to see if blockade of these would lead to the uncovering of a tonic GABAergic current due to an increase in ambient GABA concentration. In these and subsequent experiments we used BMI (20 μm) to block GABAA receptors instead of SR 95531. We chose to first block the GAT-1 GABA transporter with the blocker SKF 89976A (30 μm). GAT-1 has been reported to be present throughout most regions of the CNS in the postnatal mouse (Evans et al. 1996). In the presence of SKF 89976A alone, application of BMI did not result in a change in the holding current of HMs indicating that, with GAT-1 GABA transporters blocked, an endogenous tonic GABA current is not observed in HMs (ΔIHold= 4.4 ± 4.9 pA, n= 3, P > 0.05, data not shown). Since it has been reported that the mouse hypoglossal motor nucleus exhibits a high expression level of the GAT-3 transporter (Evans et al. 1996), we next tested whether blockade of both the GAT-1 and the GAT-3 transporter would unmask a BMI-sensitive tonic GABAA receptor-mediated current. In these experiments we blocked the GAT-3 transporter with SNAP 5114 (50 μm). Figure 1 shows that application of BMI to block GABAA receptors in the presence of blockade of both GAT-1 and GAT-3 GABA transporters did not result in an endogenous GABA current in a HM, although phasic GABAA receptor-mediated inhibitory postsynaptic currents were clearly abolished by application of the BMI. This result was observed in five HMs (ΔIHold= 2.4 ± 8.6 pA, P > 0.05).

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 GABAA receptor-mediated current in a HM.

Figure 1

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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 (IHold). Blue coloured histogram and dashed line are for the control condition (before application of BMI, mean IHold=−128.9 ± 2.9 pA) and the red coloured histogram and dashed line are after application of BMI (IHold=−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).

In the presence of exogenous GABA a tonic GABA current is observed when GABA transporters are blocked

We next tested whether application of exogenous GABA without GAT blockade would result in a GABAA receptor-mediated tonic current. In these experiments we also blocked both GABAB receptors with CGP 55845 (1 μm) and voltage-activated sodium channels with TTX (1 μm). Application of exogenous GABA (5 μm) in the absence of GABA transporter blockade did not result in unmasking of a BMI-sensitive tonic GABAA receptor-mediated current in six HMs (ΔIHold=–7.1 ± 5.4 pA, P > 0.05, data not shown).

Next we tested whether GABA transporters may be regulating extracellular GABA concentration in the presence of exogenously applied GABA. To study this, we bathed the slice in exogenous GABA (5 μm) and blocked the GAT-1 transporter with SKF 89976A. Figure 2A shows that application of exogenous GABA in the presence of the GAT-1 GABA transporter blocker did not result in the unmasking of a BMI-sensitive tonic GABAA receptor-mediated current in a HM. This result was seen in seven HMs (ΔIHold=–10.1 ± 4.3 pA, P > 0.05). Next we tested whether application of exogenous GABA (5 μm) in the presence of the GAT-3 GABA transporter blocker SNAP 5114 would result in a BMI-sensitive GABAA receptor-mediated tonic current. Figure 2B shows that application of GABA induced a BMI-sensitive inward current. Such a tonic current was seen in 11 HMs; on average the magnitude of the tonic current observed under these conditions was 18.6 ± 6.1 pA (P < 0.05).

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

Figure 2

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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 GABAA 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 GABAA 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 GABAA 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 GABAB 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 (IHold). 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 ΔIHold 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.

Recently it has been shown that, in the presence of exogenous GABA, GAT-1 and GAT-3 transporters cooperate to regulate a tonic GABA current (Gao & Smith, 2010). Therefore, we next tested whether simultaneous blockade of both transporters in the presence of exogenous GABA would result in a GABAA receptor-mediated tonic current. Figure 2C shows that application of 5 μm GABA in the presence of GAT-1 and GAT-3 blockade resulted in a robust BMI-sensitive tonic GABAA receptor-mediated current in a HM. In seven HMs the mean ΔIHold was 104.3 ± 20.9 pA (P < 0.005). Note also that the contribution of tonically activated GABAA receptors to the GABA-induced inward current is also shown by the higher baseline noise during application of GABA in the presence of the GABA transporter blockers compared with the baseline noise in the presence of BMI (see also Caraiscos et al. (2004)). Figure 2D shows the magnitude of the ΔIHold under the three different transporter blockade conditions (all in the presence of 5 μm GABA). This plot shows that the average ΔIHold in the presence of a combined blockade of GAT-1 and GAT-3 was much larger than the sum of the ΔIHold values for each transporter blocked separately.

Figure 3 shows the GABA dose–response data of the tonic current in the presence of both GABA transporter blockers. For the purpose of having as large a data set of HMs as possible from which to obtain the dose–response data we included data from experiments in which we tested whether the GABA-activated tonic current was modulated by THDOC or L-655,708 (see below). In these cases ΔIHold was measured with respect to the initial control conditions prior to application of GABA. This alternative method of calculating ΔIHold is justified because when we compared the 5 μm GABA concentration on the mean ΔIHold from GABA wash-in data (data in Fig. 3), this value did not differ statistically (unpaired t test, P > 0.2) from that found when ΔIHold was calculated as the BMI-sensitive current described in Methods (see data in Fig. 2D). Figure 3 shows that even at a GABA concentration of 1 μm there is on average a 23.8 ± 10.4 pA (n= 15 HMs) GABAA receptor-mediated tonic current. In summary, these results show that in HMs, when both GAT-1 and GAT-3 transporters are blocked, addition of GABA results in a robust dose-dependent GABAA receptor-mediated tonic current.

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

Figure 3

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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 GABAB receptors and both GAT-1 and GAT-3 transporter blockers.

GABAA receptor subunits responsible for the tonic GABA current

Tonic GABA currents have been associated with activation of GABAA receptors that contain either the α5 or the δ subunits. We used selective pharmacological agents to investigate whether or not these subunits are involved. First, we investigated whether the tonic GABAA receptor-mediated current we observed was due to activation of an α5 subunit-containing GABAA receptor. To do this we used L-655,708, which is an α5 subunit-selective partial inverse agonist. As such, if the α5 subunit were contributing to the tonic current we would anticipate that, during activation of this current, application of L-655,708 would result in a reduction of the current. As shown in Fig. 4A we found that application of L-655,708 (20 μm) did not significantly decrease the tonic BMI-sensitive current. This result in 5 μm GABA was seen in 4 of 4 HMs tested (average ΔIHold in GABA = 143.8 ± 79.2 pA, and ΔIHold in GABA + L-655,708 = 138.9 ± 77.7 pA, P > 0.05). Thus, based on this pharmacological approach, we conclude that the tonic GABAergic current does not result from activation of GABAA receptors containing the α5 subunit.

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

Figure 4

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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 GABAB 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 (IHold). 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 GABAA receptor neurosteroid modulator THDOC (500 nm) enhances the BMI-sensitive GABAA 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 GABAB 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 (IHold). 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 GABAA receptor agonist THIP activates a BMI-sensitive GABAA 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 (IHold). 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.

Next we investigated whether GABAA receptors containing the δ subunit contribute to the tonic current. First, we utilized the neurosteroid 5α-pregnane-3α,21-diol-20-one (THDOC), a positive modulator of GABAA receptors, which acts preferentially but not exclusively on δ-containing subunit assemblies of tonic GABAA receptors (Brown et al. 2002; Stell et al. 2003; Glykys & Mody, 2006). In these experiments, we applied blockers of the GAT-1 and GAT-3 GABA transporters, and then applied GABA (2 μm) to generate a tonic current. After establishment of a tonic GABA-induced current, THDOC (500 nm) was added to the perfusing fluid. If the GABAA receptor containing the δ subunit were responsible, we would anticipate that THDOC should result in an augmentation of this current. Indeed, Fig. 4B shows that this is the case. This result was seen in 5 of 5 HMs tested with THDOC. In these cells the average tonic current significantly increased by 67.7 ± 24.8 % (P < 0.05; average ΔIHold in GABA = 139.2 ± 29.8 pA, and average ΔIHold in GABA + THDOC = 222.3 ± 38.1 pA).

Second, it has been shown that the sedative and hypnotic agent 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP), also known as gaboxadol, acts as an agonist for the δ-containing subunit assembly of the tonic GABAA receptor (Brown et al. 2002; Glykys & Mody, 2006; Mortensen et al. 2010; Meera et al. 2011). In order to test whether or not THIP can activate the BMI-sensitive tonic current in HMs, we bath applied THIP (4 μm) to voltage-clamped HMs in the presence of the basic blockers of glutamatergic and glycinergic fast synaptic transmission (see above). In this case, however, we did not block the GABA transporters, GABAB receptors or add TTX to the bathing solution. Figure 4C shows that application of THIP (4 μm) alone resulted in a BMI-sensitive tonic current, which was similar to the GABA-induced BMI-sensitive tonic current we had observed. This result was seen in 9 of 9 HMs tested. In these cells the average THIP-generated tonic current was 89.0 ± 13.8 pA (P < 0.05). We also found that THIP applied at a lower concentration of 2 μm resulted in a ΔIHold of 21.5 ± 3.2 pA (P < 0.05, n= 4). Thus, based on the positive results using THDOC and THIP, we conclude that in HMs the BMI-sensitive tonic GABA current probably arises from activation of GABAA receptors containing δ subunits.

Discussion

Summary of results

In this study we describe the presence of a tonic GABAA receptor-mediated current in somatic motoneurons. This current was readily observed in the superfused brainstem slice preparation when both GAT-1 and GAT-3 GABA transporters were blocked, and the slice was also bathed in a solution containing exogenous GABA. The data also indicated that these two transporters worked in a cooperative manner to control the GABA concentration. Using a pharmacological approach we concluded that the tonic GABAergic current was probably due to activation of GABAA receptors that contain δ subunits. Based on other studies these are known to be located both peri- and extra-synaptically.

GABA transporters

In the hypoglossal motor nucleus GABA transporters function to lower the concentration of ambient GABA. Thus, in the absence of exogenous GABA the concentration is less than that needed to activate GABAA receptors responsible for the observed tonic GABA current. Our results showed that in the presence of exogenous GABA (5 μm) pharmacological blockade of GAT-1 alone did not result in a BMI-sensitive GABAA receptor-mediated tonic current. When we blocked the GAT-3 transporter alone on average a small BMI-sensitive GABAA receptor-mediated tonic current of about 20 pA was observed. In contrast, when both GAT-1 and GAT-3 were blocked, a robust average tonic current of about 100 pA (Fig. 2) was seen. This type of behaviour involving both GAT-1 and GAT-3 transporters in regulating the extracellular GABA concentration has been seen in other neural systems (Keros & Hablitz, 2005; Gao & Smith, 2010).

In mouse, four distinct GABA transporters have been described (Liu et al. 1993; Borden, 1996; Evans et al. 1996); these are mGAT-1, mGAT-2, mGAT-3 and mGAT-4. Based on sequence homology and pharmacology, the mouse mGAT-1 is similar to rat and human GAT-1, mouse mGAT-2 is similar to the dog betaine transporter (BGT), mouse mGAT-3 is similar to rat GAT-2 and mouse mGAT-4 similar to rat GAT-3 (Borden, 1996; Evans et al. 1996). Studies of the development and localization of these transporters in mouse have found that mGAT-1 mRNA is widely distributed in brain and spinal cord, mGAT-2 mRNA is found only in cerebellar granule cells, mGAT-3 mRNA is not present in brain and spinal cord and mGAT-4 mRNA is widely expressed in brain and spinal cord during the postnatal period, but in the adult mouse it has a more restricted localization (Evans et al. 1996). The GAT-1 transporter (homologous to the mGAT-1) is found mostly associated with presynaptic axon terminals, while the GAT-2 (homologous to the mGAT-3) and GAT-3 transporters (homologous to the mGAT-4) are predominantly associated with glia (Cherubini & Conti, 2001). Of particular relevance to the present study is the finding that mGAT-4 (homologous to rat GAT-3) is found in high abundance in the hypoglossal motor nucleus and spinal cord (Evans et al. 1996). The affinity of the mGAT-4 transporter for GABA is much higher than the other three GABA transporters, suggesting that it can, in the presence of sufficient numbers of transporter molecules, maintain GABA at low concentration (Liu et al. 1993). These characteristics and properties suggest an important role for the mGAT-4 (GAT-3) transporter compared with the GAT-1 transporter in regulating extracellular GABA levels in the vicinity of HMs and possibly motoneurons in general.

Regarding the selectivity of the two GAT inhibitors that we used in this study, it has been shown that SKF 89976A has high selectivity for GAT-1. SNAP 5114 has high selectivity for GAT-2 and GAT-3 versus GAT-1, with SNAP 5114 being somewhat more selective for GAT-3 versus GAT-2 (Borden, 1996). Thus, at the concentration of SNAP 5114 that we used in this study, we assume that it acts preferentially to inhibit the GAT-3 transporter.

It is of interest that in a prior study of HMs from this laboratory (Lim et al. 2004) we showed that the glycine concentration in the slice was regulated by the glycine transporter-1 (GLYT1). We found that glycine acting as a co-agonist at N-methyl-d-aspartate (NMDA) receptors was regulated by GLYT1. Thus, in the case of HMs, the effects of both inhibitory transmitters, GABA and glycine, have their availability to ligand-gated channels modulated by their respective transporters.

GABA concentration dependence

Regarding the concentration dependence of tonic current response to exogenous GABA, we observed responses at concentrations as low as 1 μm when GABA uptake was blocked. The tonic GABAA receptors containing the δ subunit (α4β3δ, α6β3δ) have been reported to have a much lower EC50 for GABA than other GABAA receptors, in the range of ∼0.3–0.7 μm (Brown et al. 2002; Wallner et al. 2003; Farrant & Nusser, 2005). For example, Burgard et al. (1996) found that the EC50 for GABA was on average between 6 and 26 μm depending on the type of β subunit for the α5βxγ2 assembly. In agreement with this, expressed α5β3γ2 GABAA receptors had an average EC50 for GABA of 19.4 μm (Caraiscos et al. 2004). Thus, the EC50 for GABA varies widely depending on the subunits that comprise the channel responsible for tonic GABA currents. In hippocampal pyramidal cells, δ subunit-containing GABAA receptor assemblies contribute to a tonic current seen in low ambient GABA concentrations, while α5 subunit-containing GABAA receptor assemblies contribute a tonic GABA current only when extracellular GABA is increased (Scimemi et al. 2005). Thus, the presence of different types of extrasynaptic GABAA receptors can expand the concentration range over which extracellular GABA exerts modulatory control of neuronal behaviour (Semyanov et al. 2004; Scimemi et al. 2005).

GABAA subunits that may be responsible for the tonic GABAergic current

There have been few studies of GABAA receptor subunit expression in rodent HMs. The early seminal work by Fritschy and Mohler (1995) found that adult rat HMs exhibited intense immunoreactivity for α1 and α2 subunits, while immunoreactivity was moderate for the γ2 and weak for the α3 and α5 subunits. More recently, and in partial agreement with this result, Lorenzo et al. (2006) found in a study of subunit immunolabelling of adult rat HMs that the α1 and α2 subunits were present, but the α3 and α5 subunits were absent from the hypoglossal nucleus. In the mouse, Muller et al. (2004) found the presence of the α1 and γ2 subunits, and these were present throughout postnatal development. While these studies focused on the subunits within the hypoglossal nucleus itself, there is no information regarding the presence of these GABAA receptor subunits in regions just outside the nucleus, regions known to contain dendrites of HMs (Altschuler et al. 1994; Nunez-Abades et al. 1994; Van Brederode et al. 2011). Regarding the expression δ subunits in HMs, to our knowledge there is no information in the mouse, and the information in the adult rat is conflicting. Using immunohistochemical methods, a report stated that cranial nerve motor nuclei lacked staining for the δ subunit (Fritschy & Mohler, 1995). However, a later report, also in the adult rat, did find staining for the δ subunit in cranial nerve motor nuclei (Pirker et al. 2000).

We used a pharmacological approach to investigate the type of GABAA receptor subunits that may be responsible for the tonic GABAergic current that was observed. The pharmacological profile that was observed is consistent with the activation of a GABAergic tonic current involving a δ subunit-containing GABAA receptor, with no or minor involvement of the α5 subunit. This is consistent with the reported absence of α5 subunit immunostaining in the XII nucleus described above.

In our study we used the α5 subunit-selective inverse agonist, L-655,708 (Bonin et al. 2007; Chen et al. 2010). If the α5 subunit-containing GABAA receptor contributed significantly to the observed tonic GABA current, then we would have anticipated to have seen an attenuation of this current with application of L-655,708 (Bonin et al. 2007; Chen et al. 2010). Our observation that there was no attenuation of the current indicates that in HMs activation of an α5 subunit-containing GABAA receptor may not contribute to the observed tonic current.

Our results showed that the neurosteroid THDOC functions as a positive modulator of tonic GABA current observed in HMs. As THDOC is known to act preferentially but not exclusively on δ-containing subunit assemblies of tonic GABAA receptors (Brown et al. 2002; Stell et al. 2003; Sciemi et al. 2005; Glykys & Mody, 2006), we conclude that activation of the δ subunit is involved in the tonic current we observed. It has been shown that THDOC potentiates the tonic GABA conductance in dentate gyrus granule cells (Stell et al. 2003), in lateral geniculate neurons (Cope et al. 2005) and in cerebellar granule cells (Stell et al. 2003). These cells have GABAA receptors with δ-containing subunits (Stell et al. 2003; Cope et al. 2005). The mechanism of action by which THDOC acts as a positive allosteric modulator of GABAA receptors is to increase their open-channel probability (Lambert et al. 2001).

Our results showed that the sedative and hypnotic agent THIP activates a BMI-sensitive tonic GABAergic current in HMs. Since it has been shown that THIP preferentially but not exclusively activates the δ subunit-containing assembly of the tonic GABAA receptor (Brown et al. 2002; Glykys & Mody, 2006; Mortensen et al. 2010; Meera et al. 2011), it is likely that this is also occurring in HMs. It has been shown in mouse and rat thalamic neurons that THIP acts on extrasynaptic δ subunit-containing GABAA receptors (Cope et al. 2005; Bright et al. 2007; Herd et al. 2009). THIP is not taken up by GABA transporters (Korn & Dingledine, 1986). Thus, from these results, we conclude that HMs possess δ subunit-containing GABAA receptors whose activation is probably responsible for the BMI-sensitive tonic GABAergic current that we have observed.

Function of tonic GABA currents

A key feature of a tonic conductance such as described here is that it can have a profound effect via shunting inhibition on excitability and what is termed ‘gain control’ of neurons (Stell et al. 2003; Bonin et al. 2007; Bright et al. 2007), as well as on network excitability (Semyanov et al. 2003). Tonic GABAA receptor activation can have an effect on input conductance of motoneurons. Thus, the effect could result in a neuron being less excitable. Recently Castro et al. (2011) provided data suggesting that adult spinal turtle motoneurons may be tonically inhibited by GABAA receptors.

There is evidence that respiratory motoneurons, whether brainstem or spinal cord, do receive GABAergic inhibition. For example, it has been shown that during inspiration, phrenic motoneurons receive concurrent glutamatergic excitation and GABAergic inhibition (Parkis et al. 1999). In vivo studies, in awake rats using microdialysis of BMI (Morrison et al. 2003), and in decerebrate dogs (Sanchez et al. 2009) using picoejection of BMI into the hypoglossal motor nucleus, have shown that endogenously active GABAA receptors inhibit inspiratory-phase-related HM activity. The most likely sites of antagonism by BMI are GABAA receptors on HMs. These studies did not investigate whether inhibition involved synaptic and/or extrasynaptic GABAA receptors, although there has been speculation that GABAergic extrasynaptic receptors are present on HMs (Donato & Nistri, 2000) and are thereby capable of producing a tonic GABA current. Another source of GABAergic inhibitory input to HMs is from Nucleus of Roller GABAergic interneurons that make monosynaptic contacts with HMs (Sumino & Nakamura, 1974). We previously observed that many of the Nucleus of Roller GABAergic cells exhibit highly regular spontaneous firing in the in vitro rhythmic slice preparation (Van Brederode et al. 2011). Thus, this activity could provide an important source of synaptically released GABA to HMs and thereby function to inhibit HMs by activation of both synaptic and by spill-over extrasynaptic GABAA receptors. The latter activation may produce a tonic GABA current such as demonstrated in this study.

Based on the current in vitro superfused slice experiments, we cannot predict whether in vivo extrasynaptic GABAA receptor-mediated tonic GABAergic inhibition of HMs is a physiological mechanism for modulation of HM excitability. In vivo conditions are clearly different from conditions in the in vitro slice, and therefore it is potentially possible that a tonic extrasynaptic GABAergic inhibition is present in vivo. In our superfused slice experiments, recordings are made from visualized HMs located superficially (generally within the top 100 μm) in the slice; thus, it is possible that the superfusion fluid could readily washout small molecules, such as GABA, that are released from synaptic sites in this superficial zone. Such washout would make it unlikely that the synaptically released GABA would spill-over to reach extrasynaptic sites in sufficient concentration to activate extrasynaptic GABAA receptors responsible for a tonic GABA current. It has been previously reported in other superfused slice preparations that in the absence of exogenous GABA a ‘tonic GABA current [was] difficult to measure’ (Zhan & Nadler, 2009). Further, Glykys and Mody (2007) in their review article on tonic GABA currents, point out that in superfused brain slice experiments, the resulting extracellular GABA concentration may be insufficient to activate GABAA receptors responsible for tonic GABA currents. Specialized anatomy, such as glia ensheathing of synapses or glomerular structures, may prevent rapid diffusion of GABA and a tonic GABA current can be observed in these cases without the need of adding exogenous GABA to the ACSF. Thus, it is not likely in the case of HMs that such specialized anatomy is present.

Additionally, extrasynaptic GABA concentration could be limited by GABA transporters whose activity may be dependent on the state of the preparation. For example, it has been shown that in some conditions GABA transporters can reverse and thereby release GABA into the extracellular space (Richerson & Wu, 2003; Wu et al. 2007). This has been shown to occur when there is elevated neuronal activity (Richerson & Wu, 2003; Wu et al. 2007) and in the pathophysiological state of ischaemia (Allen et al. 2004). Thus, in these conditions, GABA transporters could reverse and function to locally elevate GABA and thereby activate a tonic GABA current.

In conclusion, we have observed that HMs recorded in vitro exhibit a tonic GABA current that is suppressed in the in vitro slice preparation by a potent GABA transporter system. In the presence of exogenously applied GABA, simultaneous blockade of both GAT-1 and GAT-3 results in activation of a robust tonic GABA current. The tonic GABA current is not produced by activation of α5 subunit-containing GABAA receptors, but appears to be produced by activation of δ subunit-containing GABAA receptors.

Acknowledgments

This work was supported by National of Institutes of Health grant HL49657. The authors declare no conflict of interest.

Glossary

BGT

betaine transporter

BMI

bicuculline methiodide

GAT

GABA, transporter

GlyT

glycine transporter

HM

hypoglossal motoneuron

SR-95531

gabazine

THIP

gabaoxadol

Authors contributions

J.M.N. assisted in the design of the experiments, collected, analysed and aided in the interpretation of the data and revised and reviewed the manuscript. J.F.M.v.B. assisted in the design of the experiments and aided with the interpretations of the data and revised and reviewed the manuscript. A.J.B. conceived and designed the experiments, assisted in the collection, analysis and interpretation of the data. He also drafted, revised and reviewed the manuscript. All authors approved the final version.

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