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Activation of Presynaptic GABAB(1a,2) Receptors Inhibits Synaptic Transmission at Mammalian Inhibitory Cholinergic Olivocochlear–Hair Cell Synapses

  1. Eleonora Katz1,5,*
  1. 1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor N. Torres,” 1428 Buenos Aires, Argentina,
  2. 2Tercera Cátedra de Farmacología, Facultad de Medicina, 1121 Buenos Aires, Argentina,
  3. 3Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205,
  4. 4Department of Biomedicine, Institute of Physiology, University of Basel, CH-4056 Basel, Switzerland, and
  5. 5Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina

  1. Author contributions: C.W., A.B.E., and E.K. designed research; C.W., J.Z.d.S.M., J.B., M.E.G.-C., and A.V.T. performed research; C.W., P.A.F., A.B.E., and E.K. analyzed data; C.W., B.B., A.B.E., and E.K. wrote the paper.

  2. ↵*A.B.E. and E.K. contributed equally to this work.

 
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Abstract

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca2+-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca2+-activated small-conductance type 2 (SK2)K+ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABAB(1a,2) receptors [GABAB(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABABRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABAB1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABAB1a isoform selectively inhibits release at efferent cholinergic synapses.

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Introduction

In the mammalian inner ear, hair cells convert sound into electrical signals that are conveyed to the CNS via peripheral afferent neurons. In addition, hair cells and sensory neurons receive an efferent feedback from the olivocochlear (OC) system (Guinan, 2011). This system comprises the following two groups of fibers: the medial OC (MOC) fibers, whose main targets are the outer hair cells (OHCs); and the lateral OC (LOC) fibers, mainly targeting the dendrites of type I neurons that innervate inner hair cells (IHCs; Liberman et al., 1990). The MOC system is an inhibitory pathway that regulates, via the MOC–OHC synapse, the cochlear amplifier. During development, IHCs are also transiently innervated by MOC fibers (Glowatzki and Fuchs, 2000; Simmons, 2002; Katz et al., 2004; Guinan, 2011; Roux et al., 2011). Before the onset of hearing, IHCs fire spontaneous action potentials that drive the release of glutamate at the first auditory synapse (Beutner and Moser, 2001). These MOC–IHC synapses might therefore play a role in the establishment of the auditory pathway, by regulating the firing frequency of IHCs during this developmental period (Glowatzki and Fuchs, 2000; Goutman et al., 2005; Johnson et al., 2011).

At MOC–hair cell synapses, acetylcholine (ACh) activates calcium-permeable α9α10 nicotinic ACh receptors (nAChRs; Elgoyhen et al., 2001) functionally coupled to the opening of calcium-dependent small-conductance type 2 (SK2) K+ channels that hyperpolarize the hair cells (Dulon and Lenoir, 1996; Glowatzki and Fuchs, 2000; Oliver et al., 2000; Katz et al., 2011). ACh release is mediated by the activation of N- and P/Q-type voltage-gated calcium channel (VGCCs; Zorrilla de San Martín et al., 2010).

Although ACh is the main MOC neurotransmitter, a robust GABAergic innervation is seen both in the IHC and OHC areas (Fex and Altschuler, 1986; Vetter et al., 1991; Eybalin, 1993; Maison et al., 2003). Moreover, in adult mice, GABA colocalizes with ACh in almost all efferent terminals of the OC system (Maison et al., 2003). The phenotypic analysis of mice lacking GABAA receptor subunits has suggested that the GABAergic component of the OC system contributes to the long-term maintenance of hair cells and neurons in the inner ear (Maison et al., 2006). Furthermore, the analysis of GABAB1 knock-out mice has indicated that GABAergic signaling might be required for normal OHC amplifier function at low sound levels and OHC responses to high-level sound (Maison et al., 2009). In addition, GABA-mediated changes in the stiffness and motility of OHCs have been reported (Zenner et al., 1992; Batta et al., 2004). Although one study has reported that OHCs hyperpolarize in the presence of GABA (Gitter and Zenner, 1992), suggestive of the existence of postsynaptic GABA receptors, the site of action and the physiological effects of GABA at MOC–hair cell synapses are poorly understood.

To gain insight into the role of GABA in synaptic transmission at the mammalian peripheral auditory system, we searched for a postsynaptic effect of GABA at IHCs and OHCs of the developing mouse cochlea. Moreover, we studied the effect of GABABR-selective compounds on transmitter release at mouse MOC–hair cell synapses. Using pharmacological and electrophysiological approaches, together with mutant mouse lines lacking specific GABABR subtypes, we demonstrate for the first time a physiological role for GABA in OC efferent synaptic transmission. Whereas GABA does not elicit IPSCs, activation of presynaptic GABABRs inhibits the release of ACh from OC terminals. Thus, we demonstrate that the release of GABA at an inhibitory mammalian cholinergic synapse activates presynaptic GABABRs to downregulate the release of ACh.

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Materials and Methods

Animal procedures and isolation of the organ of Corti.

Procedures for preparing and recording from the postnatal mouse organ of Corti were essentially identical to those published previously (Glowatzki and Fuchs, 2000; Katz et al., 2004). Briefly, mid-apical turns of the organ of Corti were excised from BalbC GABABR mutant and control mice of either sex between postnatal day 9 (P9; day of birth was considered to be P0) and P11 for IHC recording. P12–P16 mice were used for OHC recordings. We chose P9–P11 mice for IHC recordings because ACh sensitivity has been shown to be maximal at this stage (Katz et al., 2004; Roux et al., 2011). To study the MOC–OHC synapse, we used P12–P16 mice because OHCs start to be innervated by the MOC fibers at the second postnatal week (Simmons, 2002). In addition, at P12–P16 the cochlear amplifier (OHC electromotility) is already functional (He and Dallos, 1999).

The cochlear preparations were placed in the chamber for electrophysiological recordings, mounted under an Zeiss Axioskop microscope and viewed with differential interference contrast using a 40× water-immersion objective and a camera with contrast enhancement (Dage-MTI). All experimental protocols were performed in accordance with the American Veterinary Medical Association AVMA Guidelines for the Euthanasia of Animals (June 2007).

Electrophysiological recordings.

IHCs and OHCs were identified visually by their characteristic shape and cell capacitance (7–12 pF). The cochlear preparation was continuously superfused by means of a peristaltic pump (Gilson Minipulse 3, with 8 channels, Bioesanco) containing an extracellular saline solution of an ionic composition similar to that of the perilymph as follows (in mm): 155 NaCl, 5.8 KCl, 1.3 CaCl2, 0.7 NaH2PO4, 5.6 d-glucose, and 10 HEPES buffer, pH 7.4. Working solutions containing the different drugs and toxins were made up in this same saline and delivered through the perfusion system. The pipette solution was as follows (in mm): 150 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5 HEPES buffer, and 2.5 Na2ATP, pH 7.2. Some cells were removed to access IHCs and OHCs, but mostly the pipette moved through the tissue using positive fluid flow to clear the tip. Currents in IHCs and OHCs were recorded in the whole-cell patch-clamp mode using an Axopatch 200A amplifier, low-pass filtered at 2–10 kHz and digitized at 5–20 kHz with a Digidata 1322A board (Molecular Devices). Recordings were made at room temperature (22–25°C). Glass pipettes, 1.2 mm inner diameter, had resistances of 7–10 MΩ. Recordings in OHCs were always performed at −40 mV, since at this membrane potential, stable whole-cell recordings and a good signal-to-noise ratio of the outward-going synaptic currents can be obtained (Ballestero et al., 2011). Recordings in IHCs were performed at −90 mV with the exception of those in response to exogenously applied ACh in which IHCs were voltage-clamped both at −90 and at −40 mV. An outward-going current in response to ACh is expected at −40 mV as the ACh current is composed of an inward current flowing through the α9α10 nAChRs [reversal potential (Erev) = −14 mV], plus the more prominent outward current through the Ca2+-activated SK2 potassium channel (Erev = −82 mV). At −90 mV, currents through these two channels are inward (Oliver et al., 2000; Gómez-Casati et al., 2005; Ballestero et al., 2011; Katz et al., 2011). The indicated holding potentials were not corrected for liquid junction potentials (−4 mV). Solutions containing ACh or GABA (Fig. 1) were applied by a gravity-fed multichannel glass pipette (150 μm tip diameter) positioned at ∼300 μm from the recorded IHC or OHC.

Electrical stimulation of MOC efferent axons.

Neurotransmitter release was evoked by bipolar electrical stimulation of the medial olivocochlear efferent axons as previously described (Goutman et al., 2005; Zorrilla de San Martín et al., 2010). Briefly, the electrical stimulus was delivered via a 20- to 80-μm-diameter theta glass pipette placed at 20–60 μm modiolar to the base of the recorded IHC or at the base of the IHC that was aligned with the OHC under study. The position of the pipette was adjusted until postsynaptic currents in the IHCs or OHCs (voltage-clamped at −90 or −40 mV, respectively) were consistently activated. An electrically isolated constant current source (model DS3, Digitimer Ltd) was triggered via the data acquisition computer to generate pulses up to 30 mA for 200–2000 μs.

Estimation of the quantum content of transmitter release.

The quantum content of transmitter release (m) was estimated as the ratio between the mean amplitude of evoked (eIPSCs) and the mean amplitude of spontaneous IPSCs (sIPSCs; Del Castillo and Katz, 1954). To estimate the eIPSC mean amplitude, 200 stimuli were applied at a frequency of 1 Hz. sIPSCs were recorded during and after these 200 stimuli.

The percentage of quantum content (% m) after applying a drug or toxin was calculated as follows: Formula where mc is the estimation of m in the control condition before drug application to a given cell, and mt is the quantum content estimated after incubation of the preparation with the drug or toxin under study for the time specified in the Results section (treated preparations). The variability in quantum content along the experiment in the absence of drug (considered as the control for each experiment, Control % m), was calculated comparing the quantum content at 10 min with that at 5 min after break-in for each cell and expressed as a percentage.

As the frequency of sIPSCs is extremely low in OHCs (Ballestero et al., 2011), m was calculated by the failures method (mf = ln N/N0), where N0 is the number of failures and N is the total number of successive trials (200 trials at a frequency of 1 Hz; Hubbard et al., 1969). Failures of release were computed in the absence or presence of the different drugs or toxins.

Statistical significance was evaluated by paired Student's t test (one-tailed) unless otherwise stated. A p < 0.05 was considered significant. All data were expressed as the mean ± SEM. Synaptic currents were analyzed with Minianalysis (Synaptosoft) and Clampfit 9.2 (Molecular Devices).

Cochlear immunohistochemistry.

Whole cochleae were dissected and immediately perfused through the round window with 4% paraformaldehyde (PFA) in PBS, pH 7.4. Cochleae were fixed in 4% PFA overnight at 4°C before rinsing with PBS. Apical turns of the organs of Corti were excised from the cochleae and blocked in blocking buffer (PBS with 5% normal goat serum and 0.3% Triton X-100) for 2 h at room temperature. Cochlear apical turns were incubated with the primary antibody diluted in blocking buffer overnight at 4°C, rinsed three times for 20 min in PBT (PBS with 0.3% Triton X-100), incubated with the secondary antibody diluted in blocking buffer overnight at 4°C, rinsed three times for 20 min in PBT, and rinsed in PBS before mounting on glass slides in Vectashield mounting medium (Vector Laboratories). A laser scanning confocal microscope (Fluoroview, Olympus) was used to acquire images of the whole mounted organs of Corti.

The following antibodies were used: rabbit anti-GFP (1:1000; Invitrogen), rabbit anti-glutamic acid decarboxylase (GAD; 1:500; Synaptic Systems), mouse anti-synaptophysin (1:1000; Millipore), and secondary Alexa Fluor 488- and 568-labeled antibodies (1:1000; Invitrogen).

Drugs and toxins.

Stock solutions of peptide toxins were prepared in water. Drugs and reagents were from Sigma-Aldrich, ω-conotoxin-GVIA was from Alomone Labs; and ω-agatoxin-IVA (ω-AgaIVA) was from Peptides International Inc.. All drugs and toxins were thawed and diluted in the extracellular solution shortly before use.

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Results

Effects of exogenously applied GABA to OHCs and IHCs

We first evaluated whether GABA is able to elicit postsynaptic currents in mouse cochlear hair cells. GABA (1 mm) was applied, via a gravity-fed perfusion system, to voltage-clamped IHCs and OHCs in the cochlear preparation. As a positive control, we applied 1 mm ACh to all evaluated cells (Fig. 1).

Figure 1.
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Figure 1.

Effects of exogenously applied GABA to cochlear hair cells. A, Representative records obtained in IHCs at P9–P11 upon application of GABA and ACh; 1 mm GABA failed to evoke currents at either −40 or −90 mV in all IHCs tested. ACh, used as a positive control, evoked currents at both potentials in all evaluated IHCs. B, Representative records obtained in OHCs at P12–P14 upon application of GABA and ACh. In OHCs, 1 mm GABA failed to evoke currents at −40 mV in all cells tested, whereas ACh evoked currents in all evaluated cells. (Note that OHCs were voltage-clamped at −40 mV and IHCs at both −90 and −40 mV; see Materials and Methods for a detailed explanation of ACh-evoked currents.)

In P9–P11 IHCs, the age within the short period at which these cells are innervated by MOC efferent fibers (Glowatzki and Fuchs, 2000; Simmons, 2002; Katz et al., 2004; Guinan, 2011; Roux et al., 2011) and at which both the number of functionally innervated cells and their sensitivity to ACh are maximal (Katz et al., 2004; Roux et al., 2011), 1 mm GABA failed to elicit a postsynaptic current (n = 3 cells, two mice; Fig. 1A) when voltage-clamped at −40 mV. As under our recording conditions the equilibrium potential for Cl− (ECl−) is 0 mV, we also tested the effects of GABA at −90 mV, which would enhance a potential current carried by chloride ions. As shown in the bottom of Figure 1A, GABA also failed to elicit currents in IHCs voltage-clamped at this potential. In the same cells 1 mm ACh evoked robust currents at the two holding potentials tested (226.6 ± 50.2 pA at −40 mV, n = 3; and −231.7 ± 18.8 pA at −90 mV, n = 4; Fig. 1A).

OHCs (P12–P16), the final targets of MOC efferent innervation (Simmons et al., 1996; Simmons, 2002), also lacked responses to 1 mm GABA when voltage-clamped at −40 mV. In the same cells, 1 mm ACh consistently evoked robust outward currents (129.0 ± 21.84 pA; n = 15 cells, 12 mice; Fig. 1B). The effects of GABA in OHCs were only tested at −40 mV because these cells became very leaky and unstable at −90 mV (Ballestero et al., 2011). These results clearly show that IHCs and OHCs, at ages at which they are functionally innervated by MOC efferent fibers, lack any GABAA receptor-mediated responses.

Transmitter release at the MOC–IHC synapse is modified by GABAB receptor compounds

In view of the lack of postsynaptic responses to exogenously applied GABA in hair cells, we asked whether GABA activates presynaptic GABABRs to inhibit ACh release. Thus, we tested the effects of GABABR-selective drugs on the quantum content of transmitter release. Cholinergic postsynaptic responses evoked with single shocks, at a stimulation frequency of 1 Hz, were measured in P9–P11 IHCs voltage-clamped at −90 mV. The quantum content of evoked release (m) was obtained by calculating the mean eIPSC amplitude in every cell and dividing it by the mean sIPSC amplitude (Del Castillo and Katz, 1954). Under our recording conditions, m was 1.2 ± 0.2 (n = 22 cells, 21 mice), a value similar to that reported previously in IHCs from both rat (Goutman et al., 2005) and mouse (Zorrilla de San Martín et al., 2010) cochlear preparations. The quantum content of evoked release, evaluated by 200 stimuli given at 1 Hz each 5 min, remained constant during at least 60 min (data not shown). A 5–7 min incubation of the cochlear preparation with 1 μm baclofen, a selective GABABR agonist (Misgeld et al., 1995), significantly reduced the amplitude of eIPSCs (control: 39.7 ± 6.1 pA, 1192 events, 8 cells, 6 mice; baclofen: 19.4 ± 2.4 pA, 614 events, 8 cells, 7 mice; unpaired Student's t test, p < 0.01; Fig. 2A), without affecting the amplitude of sIPSCs (control: 11.0 ± 0.9 pA, 1651 events; baclofen: 12.0 ± 1.6 pA, 759 events, p = 0.24; Fig. 2A, right, inset). Under these conditions, m was reduced to 74.4 ± 6.9% and 57.07 ± 8.3% of the initial value (p < 0.05), in the presence of 0.3 and 1 μm baclofen, respectively [control: 126.6 ± 12.3% for 0.3 μm baclofen, 6 cells, 6 mice; and 91.2 ± 7.1% for 1 μm baclofen, 8 cells, 7 mice; Fig. 2B (the bars for the respective control groups are not shown); see Materials and Methods for explanation of how % m was calculated for both control and treated preparations]. As illustrated in Figure 2C, baclofen significantly reduced m in the eight cells tested (control: 1.53 ± 0.32; 1 μm baclofen: 0.87 ± 0.23; p < 0.01).

Figure 2.
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Figure 2.

Baclofen, a specific GABABR agonist, reduces the quantum content of eIPSCs at the MOC–IHC synapse. A, Representative traces of eIPSCs recorded at a holding potential of −90 mV before (left) and after 5 min of incubation with 1 μm baclofen (right). The inset in A (right) shows that 1 μm baclofen (gray) did not affect the amplitude of sIPSCs. B, Bar graph showing that baclofen (0.3 and 1 μm) caused a significant reduction in m. Results are expressed as a percentage of control. C, Summary plot for individual cells recorded before (open circles) and after 5 min of incubation with 1 μm baclofen (filled circles). The mean ± SEM values obtained before and after incubation with baclofen are plotted to the left and to the right of their respective individual responses. *p < 0.05, **p < 0.01, paired Student's t test.

These results suggest that GABA regulates the amount of ACh released at the MOC–IHC synapse through the activation of GABABR. To further test this hypothesis, we studied the effects of CGP35348, a selective GABABR antagonist (Bittiger et al., 1993; Fig. 3). CGP35348, at a concentration of 1 μm, significantly increased the amplitude of eIPSCs after 5–7 min of incubation (control: 25.4 ± 11.6 pA, 617 events; CGP35348: 28.5 ± 5.9 pA, 664 events, 6 cells, 6 mice; p < 0.05; Fig. 3A) without affecting the amplitude of sIPCS (control: 14.85 ± 0.49 pA, 558 events; CGP35348: 13.5 ± 0.93 pA, p = 0.08, 311 events; Fig. 3A, right, inset). Consistent with this result, and in agreement with a presynaptic site of action of CGP35348, m was significantly increased by 1 μm CGP35348 (control: 101.5 ± 12.9%; CGP35348: 147.7 ± 18%; 6 cells, 6 mice; p < 0.05; Fig. 3B). The summary plots in Figure 3C show the six cells tested, where 1 μm CGP35348 significantly increased m (control: 1.02 ± 0.41; CGP35348: 1.3 ± 0.45; 6 cells, 6 mice; p < 0.05). The fact that both baclofen and CGP35348, the GABABR agonist and antagonist, respectively, significantly modified the quantum content of evoked release, strongly suggests that GABA inhibits the release of ACh through the activation of presynaptic GABABR.

Figure 3.
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Figure 3.

CGP35348, a specific GABABR antagonist, increases the quantum content of eIPSCs at the MOC–IHC synapse. A, Representative traces of eIPSCs recorded at a holding potential of −90 mV before (left) and after 5 min of incubation with 1 μm CGP35348 (right). The inset in A (right) shows that 1 μMCGP35348 (gray) did not affect the amplitude of sIPSCs. B, Bar graph showing that CGP35348 caused a significant enhancement in m only at a concentration of 1 μm. Results are expressed as a percentage of control. C, Summary plot for individual cells recorded before (open circles) and after 5 min of incubation with 1 μm CGP35348 (filled circles). The mean ± SEM values of m obtained before and after incubation with GCP35348 are plotted to the left and to the right of their respective individual responses. *p < 0.05, paired Student's t test.

Baclofen failed to modify evoked transmitter release at the MOC–IHC synapse in mice lacking functional GABABRs

To further explore the involvement of GABABR in the release of ACh from MOC efferent fibers contacting IHCs, we studied the effects of baclofen in GABAB1 knock-out (1−/−) mice lacking functional GABABRs (Schuler et al., 2001; Bettler et al., 2004; Gassmann et al., 2004; Gassmann and Bettler, 2012). Application of 1 μm baclofen significantly reduced m in wild-type (1+/+) mice (Fig. 4A, left; control: 109.4 ± 10.4%; baclofen: 83.3 ± 9.2%; 6 cells, 6 mice; p < 0.05), but not in 1−/− littermate mice (Fig. 4B, left; control: 99.0 ± 6.5%; baclofen: 93.8 ± 8.1%; 6 cells, 6 mice; p = 0.32). No significant differences were found in either sIPSC or eIPSC amplitude between wild-type and 1−/− mice (data not shown). The summary plots for individual cells illustrate that 1 μm baclofen significantly reduced m in 1+/+ mice (control: 1.63 ± 0.3; baclofen: 1.33 ± 0.21; 6 cells; p < 0.05; Fig. 4A, right). However, baclofen did not modify transmitter release (control: 1.04 ± 0.20; baclofen: 0.95 ± 0.16; 6 cells; p = 0.13) in 1−/− mice (Fig. 4B, right). This result further supports the hypothesis that GABA inhibits transmitter release at the MOC–IHC synapse by acting through presynaptic GABABRs.

Figure 4.
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Figure 4.

Baclofen fails to reduce the quantum content of eIPSCs at the MOC–IHC synapse in mice lacking functional GABABRs. A, B, Left, Bar graphs showing that 1 μm baclofen reduced m in wild-type mice (1+/+, A) but not in mice lacking functional GABABRs (1−/−, B). Results are expressed as a percentage of control. A, B, Right, Summary plots for individual cells recorded before (open circles) and after 5 min of incubation with 1 μm baclofen (filled circles) in 1+/+ (A) and 1−/− mice (B). The mean ± SEM values of m obtained before and after incubation with baclofen are plotted to the left and to the right of their respective individual responses. *p < 0.05 paired Student's t test.

GABA inhibits the release of ACh at the MOC–IHC synapse through presynaptic GABAB(1a,2)Rs

Functional GABABRs are heterodimers composed of the GABAB1 and GABAB2 subunits (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999). Molecular diversity of GABABR arises from two different and pharmacologically indistinguishable GB1 isoforms, 1a and 1b (Bettler et al., 2004). There is evidence indicating that these two isoforms are expressed at different synaptic sites, with GB1a predominantly located at the presynapse and GB1b at the postsynapse (Pérez-Garci et al., 2006; Vigot et al., 2006).

To determine which GB1 isoform is involved in the inhibition of ACh release at the MOC–IHC synapse, we compared the effects of baclofen in mutant mice lacking either the GABAB1a (1a−/−) or the GABAB1b (1b−/−) isoform (Vigot et al., 2006; Fig. 5). Application of 1 μm baclofen significantly reduced m at MOC–IHC synapses from wild-type littermate mice of both isoforms (1a+/+: Fig. 5A, left; control: 94.3 ± 10.1%; baclofen: 64.6 ± 8.8%; 10 cells, 8 mice; p < 0.05; 1b+/+: Fig. 5C, left; control: 102.5 ± 8.1%; baclofen: 72.9 ± 4.2%; 7 cells, 7 mice; p < 0.01). Baclofen did not modify m in 1a−/− mice (Fig. 5B, left; control: 106.3 ± 7.0%; baclofen: 96.9 ± 6.8%; 13 cells, 9 mice; p = 0.09), indicating that the GABAB1a isoform inhibits transmitter release. On the contrary, m was significantly reduced in 1b−/− mice, excluding an involvement of the GABAB1b isoform (Fig. 5D, left; control: 113.4 ± 10.5%; baclofen: 70.7 ± 10.0%; 7 cells, 6 mice; p < 0.01). The right panels in Figure 5A–D are the summary plots for individual cells from each group of mice recorded in control conditions and after 7 min of incubation with 1 μm baclofen (Fig. 5A, 1a+/+: control, 1.60 ± 0.2; baclofen, 1.04 ± 0.21; 10 cells; p < 0.01; Fig. 5B, 1a−/−: control, 1.56 ± 0.25; baclofen, 1.54 ± 0.27; 13 cells; p = 0.46; Fig. 5C, 1b+/+: control, 1.33 ± 0.2; baclofen, 0.92 ± 0.1; 7 cells; p < 0.01; Fig. 5D, 1b−/−: control, 1.5 ± 0.3; baclofen, 1.03 ± 0.27; 7 cells; p < 0.01). Baclofen did not alter the amplitude of sIPSCs in any of the GABABR knock-out mice (data not shown). Together, our results strongly suggest that ACh release at the MOC–IHC synapse is inhibited by GABA acting on presynaptic GABAB1a-containing receptors, the GABAB(1a,2)Rs.

Figure 5.
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Figure 5.

Baclofen fails to reduce the quantum content of eIPSCs at the MOC–IHC synapse in mice lacking the 1a GABABR isoform. A–D, Quantum content of eIPSC recorded in IHCs, evoked by electrical stimulation of the MOC efferent axons before and after incubation with 1 μm baclofen in wild-type (A, C) and in isoform-specific GABAB knock-out mice: 1a−/−) in B; 1b+/+ in D. In the bar graphs, results are expressed as a percentage of control. Summary plots for individual cells recorded before and after 7 min of incubation with 1 μm baclofen are shown on the right in A–D. The mean ± SEM values of m obtained before and after incubation with baclofen are plotted to the left and to the right of their respective individual responses. *p < 0.05, **p < 0.01, paired Student's t test.

P/Q-type VGCCs are inhibited following GABABR activation

We have recently shown that both P/Q- and N-type VGCCs support neurotransmitter release at the mouse MOC–IHC synapse before the onset of hearing (Zorrilla de San Martín et al., 2010). To gain insight into the mechanisms underlying the inhibition that GABA exerts on ACh release at this synapse, we tested the effects of the GABAB antagonist CGP35348 in the presence of blockers of P/Q- and N-type VGCCs, ω-AgaIVA, and ω-conotoxin GVIA (ω-CgTx), respectively.

As previously reported (Zorrilla de San Martín et al., 2010), m was greatly reduced after 15 min of incubation with 200 nm ω-AgaIVA (Fig. 6A,B; control: 1.20 ± 0.14; 9 cells, 9 mice; ω-AgaIVA: 0.26 ± 0.1; 11 cells, 9 mice; p < 0.001). Under this condition, CGP35348 failed to significantly increase the amount of transmitter released by the MOC fibers (Fig. 6A,B; +CGP: 0.34 ± 0.1; 9 cells, 9 mice; p = 0.07). The inset in Figure 6 shows representative averaged traces illustrating the effects of ω-AgaIVA and CGP35348 on the amplitude of eIPSCs. sIPSCs were not affected by either of these drugs (data not illustrated; control: 9.33 ± 0.4 pA; ω-AgaIVA: 8.6 ± 0.3 pA; +CGP: 8.4 ± 0.4 pA; 9 cells, 9 mice; ANOVA, p = 0.17; n = 179, 123, and 104 events, respectively).

Figure 6.
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Figure 6.

P/Q-type Ca2+channels are the target of GABAB receptor activation at the transient MOC–IHC synapse. A, B, Bar diagram (A) and the plot of individual cells (B) show the effects of 1 μm CGP35348 on m after blocking P/Q-type VGCCs with 200 nm ω-agatoxin-IVA. Inset between A and B illustrates representative averaged traces showing the effects of CGP on the amplitude of eIPSPs in the presence of ω-agatoxin-IVA (control, black; ω-Aga, gray; +CGP, red). C, D, Bar diagram (C) and plot of individual cells (D) illustrating the effect of 1 μm CGP35348 on transmitter release after blocking N-type VGCCs with 500 nm ω-conotoxin GVIA. Inset between graphs illustrates representative averaged traces showing the effects of CGP35348 on the amplitude of eIPSPs in the presence of ω-conotoxin GVIA (control, black; ω-CgTx, gray; +CGP, red). Red horizontal bars in B and D represent the mean in each group. Error bars represent the SEM. *p < 0.05, ***p < 0.001, paired Student's t test.

Figure 6D and E, shows that m was, as previously reported (Zorrilla de San Martín et al., 2010) significantly reduced after 15 min of incubation with 500 nm ω-CgTx (control: 0.73 ± 0.1; 7 cells, 7 mice; ω-CgTx; 0.35 ± 0.1; 13 cells, 11 mice; p < 0.001). Under this condition, 1 μm CGP35348 was still able to significantly increase m when compared with the ω-CgTx-treated group (+CGP: 0.51 ± 0.1; 10 cells, 8 mice; p < 0.05). Figure 6, inset, shows representative averaged traces illustrating the effect of ω-CgTx and CGP35348 on the amplitude of eIPSPs. sIPSC amplitude was not affected by ω-CgTx or ω-CgTx+CGP (data not illustrated; control: 11.1 ± 0.2 pA; ω-CgTx: 11.5 ± 0.3 pA; +CGP: 10.9 ± 0.2 pA; 7 cells, 7 mice; ANOVA, p = 0.21, n = 300, 210, and 185 events, respectively). Together, these results suggest that the activity of P/Q-type VGCCS is reduced following GABABR activation.

GABA regulates the release of ACh at the MOC–OHCs synapse through inhibitory presynaptic GABABRs

At the beginning of the second postnatal week, OHCs begin to respond to ACh (Dulon and Lenoir, 1996; He and Dallos, 1999). To determine whether GABA exerts an inhibitory effect on ACh release at these synapses, we tested the effects of the GABABR antagonist CGP35348 on the quantum content of evoked release at the MOC–OHC synapse (Fig. 7). As the frequency of sIPSCs at MOC–OHC synapses is very low, m was evaluated by the failures method (see Materials and Methods).

Figure 7.
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Figure 7.

CGP35348 increases the quantum content of transmitter release at the MOC–OHC synapse. A, B, Representative traces of eIPSCs recorded in OHCs at −40 mV, before and after incubation with 1 μm CGP35348. C, Bar diagram showing that CGP35348 (1 μm) significantly increased m at the MOC–OHC synapse. Results are expressed as a percentage of control. D, Summary plot for individual cells recorded before and after 15 min of incubation with 1 μm CGP35348. The mean ± SEM values of m obtained before and after incubation with CGP35348 are plotted to the left and to the right of their respective individual responses. *p < 0.05, **p < 0.01, paired Student's t test.

Representative traces of eIPSCs recorded in OHCs (P14) before (Fig. 7A) and after (Fig. 7B) incubating the cochlear preparation with 1 μm CGP35348 show that the number of failures decreased in the presence of the antagonist. Consistently, ACh release was significantly enhanced in the presence of the antagonist (Fig. 7C; control: 111.0 ± 26.6%; CGP35348: 170.4 ± 37.5%; 4 cells, 3 mice; p < 0.05). The summary plot in Figure 7D shows that m significantly increased in the four cells tested after incubating the cochlear preparation with CGP35348 (control: 0.12 ± 0.02; CGP35348: 0.29 ± 0.04; p < 0.01). These results indicate that GABA inhibits ACh release at the MOC–OHC synapse through the activation of presynaptic GABABRs. In a future work, experiments will be carried out to test whether ACh release at the MOC–OHC synapse is also inhibited by GABA acting on presynaptic GABAB(1a,2)Rs.

GABABRs are expressed in efferent synaptic terminals at the base of IHCs and OHCs before the onset of hearing

The electrophysiological experiments shown above demonstrate that synaptic activation of GABABRs inhibits ACh release at both MOC–IHC and MOC–OHC synapses. To confirm the expression of GABABRs at the terminals of these synapses, we used a transgenic mouse line (GABAB1-GFP) in which GFP is fused to the GABAB1 subunit (Casanova et al., 2009; Fig. 8). Cochlear whole-mount preparations from P9–P14 were coimmunostained with GFP antibodies to enhance the endogenous GFP signal and to detect GFP-tagged GABAB1 subunits and synaptophysin antibodies to identify efferent terminals contacting hair cells. A GABAB1-GFP-positive signal was detected in the inner spiral bundle region at the base of IHCs containing both efferent and type I afferent neurons. A strong colocalization of GFP with synaptophysin was observed, indicating the presence of GABABRs at efferent terminals under the IHCs (Fig. 8A–C). In the OHC region, GABAB1 expression was found both in type II afferent and MOC efferent fibers contacting the OHCs (Fig. 8C, inset, filled arrow). The expression of GABABRs at MOC terminals suggests the presence of the neurotransmitter GABA at the synapse. To study whether GABA is present at efferent terminals contacting hair cells at ages around the onset of hearing, we used an antibody that recognizes the GABA synthetic enzyme GAD (Fig. 8D–F) together with an antibody recognizing synaptophysin. In regions below IHCs and OHCs, GAD colocalized with synaptophysin (Fig. 8F, inset, filled arrow). These immunostainings are consistent with our electrophysiological results showing that GABA is involved in the modulation of synaptic transmission at MOC–hair cell efferent synapses during development.

Figure 8.
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Figure 8.

GABABRs are expressed in efferent synaptic terminals at the base of IHCs and OHCs. A–C, Cochlear whole-mount preparations from P9 to P14 double-immunostained with anti-GFP; A, green), and synaptophysin (Syn; B, red). A strong colocalization of GFP with synaptophysin can be observed (C, yellow) in the inner spiral bundle region at the base of IHCs (efferent and type I afferent neurons) and, at the OHC region, in type II afferent and MOC efferent fibers contacting the cells (C, filled arrow). D–F, cochlear preparations from similar aged mice immunostained with an antibody to the GABA synthetic enzyme GAD (D, green) together with synaptophysin (E, red). F, Immunoreactivity is visible as small puncta below IHCs (filled arrow) and OHCs indicating that GAD colocalized with synaptophysin at these regions (yellow). ISN, Inner spiral bundle; OSB, outer spiral bundle.

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Discussion

Lack of GABA postsynaptic effects

The present results show that GABA failed to elicit postsynaptic currents in either OHCs or IHCs. This suggests that, at least in mid-apical cochlear turns from P9–P16 mice, there are no functional postsynaptic GABAA receptors at either the MOC–OHC or the MOC–IHC synapse. The discrepancy with a report showing that application of GABA hyperpolarizes OHCs (Gitter and Zenner, 1992) might be explained by differences in species (guinea pigs vs mice) and/or age (adult vs P12–P16) of the animals used in the experiments. The absence of postsynaptic GABAA-mediated responses is consistent with the observation that when the α9α10 nAChR is pharmacologically blocked, no postsynaptic currents in response to either K+ elevation or electrical stimulation of the MOC efferent axons are seen in OHCs (Oliver et al., 2000; Ballestero et al., 2011) and IHCs (Glowatzki and Fuchs, 2000). Moreover, it is in line with the finding that in α9 knock-out mice, no postsynaptic currents are left (Vetter et al., 2007), clearly indicating that fast synaptic transmission at the OC–hair synapse is cholinergic and mediated only through the α9α10 nAChR (Fig. 9). Moreover, the fact that GABAB receptors are not expressed either in postnatal (present work) or adult hair cells (Maison et al., 2009) further suggests a lack of a postsynaptic GABAB-mediated action. Together, these results support the idea that the main effect of the profuse OC GABAergic innervation that makes direct synaptic contacts with cochlear hair cells is not a postsynaptic one.

Figure 9.
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Figure 9.

Schematic representation of the ion channels and receptors that support and regulate transmitter release from the MOC efferent synaptic terminals. After invasion of the terminal action potential, Ca2+ entering through both P/Q- and N-type VGCCs supports the release of ACh, while L-type VGCCs exert a negative control on this process by activating BK channels (Zorrilla de San Martín et al., 2010). The present results strongly suggest that GABA, coreleased with ACh, activates presynaptic GABAB(1a,2)Rs, which negatively regulate the release of ACh by altering Ca2+ influx through P/Q-type VGCCs.

GABA modulates transmitter release at MOC–hair cell synapses through GABAB(1a,2)Rs

The results obtained with the GABABR agonist and antagonist indicate that GABA inhibits ACh release at MOC–hair cell synapses through presynaptic GABABRs. These results are in agreement with the classical role of GABA in presynaptic modulation of synaptic transmission at mammalian glutamatergic and GABAergic synapses via heteroreceptors and autoreceptors, respectively (Gaiarsa et al., 1995; Brenowitz et al., 1998; Chalifoux and Carter, 2011; Fischl et al., 2012). The modulation of ACh release, as described in the present work, although not widely observed, is not without precedent. For example, the activation of presynaptic GABABRs inhibits ACh release from Caenorhabditis elegans motoneurons (Schultheis et al., 2011) and from cholinergic nerve terminals in the rat superior cervical ganglion (Farkas et al., 1986). However, the present results are unique due to the inhibitory sign of the cholinergic OC–hair cell synapse. Thus, the release of GABA at the inhibitory mammalian cholinergic synapse activates presynaptic GABABRs that inhibit the release of ACh and therefore curtail inhibition.

The lack of effect of baclofen in mice with a specific ablation in the GB1a (1a−/−) subunit isoform (Vigot et al., 2006) clearly demonstrates the involvement of the GB1a isoform in the presynaptic inhibition of ACh release at the MOC–IHC synapse. This is in agreement with the established role of GABAB1a in presynaptic inhibition (Gassmann and Bettler, 2012). For example, at glutamatergic hippocampal CA3–CA1 synapses, GABAB1a mainly assembles into presynaptic heteroreceptors, while receptors formed by the GABAB1b subunit mediate postsynaptic inhibition (Vigot et al., 2006). Similarly, presynaptic inhibition at GABAergic synapses between synaptic terminals from neurons in cortical layer 1 and pyramidal neurons in cortical layer 5 is absent in the 1a−/− but not in the 1b−/− mice, suggesting that GABAB1a but not GABAB1b subunits assemble into presynaptic autoreceptors (Pérez-Garci et al., 2006). Our findings at a cholinergic synapse thus provide further evidence for distinct physiological roles and a differential distribution of GABAB1 subunit isoforms.

GABABRs negatively regulate synaptic transmission at the MOC–IHC synapse by inhibiting P/Q-type VGCCs

At the MOC–IHC synapse, ACh release is mediated by both N- and P/Q-type VGCCs (Zorrilla de San Martín et al., 2010). At several central synapses, presynaptic GABABRs reduce transmitter release by direct inhibition of P/Q- and N-type VGCCs through Gβγ subunits of the activated G-protein (Mintz and Bean, 1993; Thompson et al., 1993; Lambert and Wilson, 1996; Bowery et al., 2002; Bettler et al., 2004). The fact that the GABABRs antagonist CGP35348 failed to significantly increase transmitter release at the MOC–IHC synapse when P/Q-type VGCCs were completely blocked by ω-agatoxin-IVA, together with the observation that CGP353548 increased transmitter release after blocking N-type VGCCs with ω-conotoxin GVIA, indicates that P/Q- and not N-type VGCCs are inhibited by GABABR activation (Fig. 9). However, our results do not exclude the participation of other proteins in the GABAB signaling cascade, like L-type VGCCs (Shen and Slaughter, 1999; Zorrilla de San Martín et al., 2010; Bray and Mynlieff, 2011) and like proteins of the vesicular release machinery downstream of VGCCs (Yoon et al., 2007).

GABABRs are expressed in efferent synaptic terminals at the base of IHCs and OHCs

The electrophysiological data suggest the presence of functional GABABRs at MOC synaptic terminals contacting both the IHCs and OHCs. Immunostaining experiments in transgenic GABAB1-GFP mice further demonstrate the expression of GABABRs in OC terminals contacting these cells. In the IHC area, the GABAB1-GFP immunostaining arising from the OC terminals could, in principle, derive from the LOC fibers contacting type I afferent dendrites, the MOC fibers that transiently innervate the IHCs, or both (Liberman et al., 1990; Vetter et al., 1991; Simmons, 2002; Maison et al., 2003). Our electrophysiological data, however, rule out the possibility that GABABRs are solely expressed in the LOC fibers innervating the afferent neurons and clearly demonstrate that functional GABABRs are present at the MOC synaptic terminals making axo-somatic synapses with IHCs.

The lack of GFP immunostaining in OHCs and IHCs, and the presence of GFP immunostaining in type I and type II afferent fibers in GABAB1-GFP mice during development replicate the findings that were made with adult mice (Maison et al., 2009). In addition, this agrees with the presence of GABABR transcripts in mouse spiral ganglion neurons from embryonic through adult stages (Lin et al., 2000). Interestingly, in adult mice, GABABRs were not detected in OC efferent terminals in either the IHC or the OHC areas (Maison et al., 2009). Together, the present results and those of Maison et al. (2009) suggest that the OC efferent synaptic terminals transiently express GABABRs. Moreover, the results imply that GABABRs are expressed in type I and type II afferent fibers before the onset of hearing and remain there through adulthood. In contrast, GABABRs expressed in OC terminals are downregulated between the onset of hearing and adulthood. Downregulation of GABABR during development, most prominently the GABAB1a isoform, has been described (Malitschek et al., 1998; Bianchi et al., 2005). Interestingly, the expression of two postsynaptic key molecules of the transient MOC–IHC synapse, the α10 nAChR subunit and the SK2 K+ channel, also cease to express around the onset of hearing (Katz et al., 2004), further highlighting the importance of cochlear synaptic refinement during the critical period (Simmons et al., 1996).

Functional consequences

Our functional data, together with the immunodetection of GABAB receptors at OC terminals and of GAD in fibers reveal the existence of GABAergic OC fibers contacting IHCs and OHCs around the onset of hearing. These results are in agreement with the notion that the only source of GABAergic input to the mammalian cochlea is the OC system (Fex and Altschuler, 1986; Thompson et al., 1986; Vetter et al., 1991; Eybalin, 1993; Maison et al., 2003). Moreover, our functional data suggest that GABA and ACh are both released upon MOC fiber activity (Fig. 9). Based on the observations that in adult mice GABA and ACh are colocalized in the same terminals (Maison et al., 2003), we hypothesize that both neurotransmitters are coreleased upon efferent activation. Corelease of GABA and ACh, two fast neurotransmitters of usually opposite excitability, from the same terminal has been described for the retinal starburst amacrine cells (Duarte et al., 1999; Lee et al., 2010), where both neurotransmitters act at postsynaptic receptors. However, a different scenario is observed at the OC–hair synapse, where ACh release might be regulated by coreleased GABA acting at GABAB autoreceptors.

Presynaptic inhibition of ACh release via GABABRs might shape short-term plasticity properties of the MOC–hair cell synapses by regulating the release probability and, thus, as reported for other synapses (Brenowitz et al., 1998; Brenowitz and Trussell, 2001), allow continuous signaling upon high-frequency MOC activity. Since MOC firing frequency increases with sound intensity (Robertson and Gummer, 1985; Brown, 1989) and the efficacy of the MOC–IHC synapse is optimal when MOC fibers are activated at high frequency (Ballestero et al., 2011), presynaptic inhibition might therefore be relevant for MOC protection from acoustic trauma (Rajan, 2000; Taranda et al., 2009).

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Conclusions

Synaptic strength is a key variable during development for both establishing and refining connections. Our results show that during the development of the auditory system GABA inhibits the amount of ACh released at the MOC–hair cell synapse through presynaptic GABAB(1a,2)Rs, coupled to P/Q-type VGCCS. Thus, we demonstrate that GABA regulates the strength of the MOC–IHC synapse, and could thereby control the IHC firing frequency, and, therefore, the release of glutamate in the first auditory synapse at a critical period where synaptic connections are actively being formed in the cochlea, also a period where the entire auditory pathway is being established.

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Footnotes

    • Received June 17, 2013.
    • Revision received July 31, 2013.
    • Accepted August 20, 2013.

  • This work was supported by research grants from the University of Buenos Aires to E.K. (UBACYT 2008-10; 2011-14) and A.B.E. (UBACYT 2008-2010); from Agencia Nacional de Promoción Científica y Tecnológica (Argentina) to A.B.E. and E.K.; from Howard Hughes Medical Institute International Scholars Program to A.B.E.; from National Institutes of Health (R01 DC001508) to P.A.F. and A.B.E.; from the National Organization for Hearing Research to M.E.G.-C.; and from the Swiss National Science Foundation (3100A0-117816) to B.B.

  • Correspondence should be addressed to Eleonora Katz, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina. eleokatz{at}gmail.com
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  1. The Journal of Neuroscience, 33(39): 15477-15487; doi: 10.1523/JNEUROSCI.2554-13.2013
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