Functional roles of high-affinity glutamate transporters in cochlear afferent synaptic transmission in the mouse

Abstract

In the cochlea, afferent transmission between inner hair cells and auditory neurons is mediated by glutamate receptors. Glutamate transporters located near the synapse and in spiral ganglion neurons are thought to maintain low synaptic levels of glutamate. We analyzed three glutamate transporter blockers for their ability to alter the effects of glutamate, exogenously applied to the synapse via perfusion of the scala tympani of the mouse, and compared that action to their ability to alter the effects of intense acoustic stimulation. Threo-beta-benzyloxyaspartate (TBOA) is a broad-spectrum glutamate transporter antagonist, affecting all three transporters [glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT1), and excitatory amino acid carrier 1 (EAAC1)]. l-serine-O-sulfate (SOS) blocks both GLAST and EAAC1 without effect on GLT1. Dihydrokainate (DHK) is selective for GLT1. Infusion of glutamate (10 microM for 220 min), TBOA (200 microM for 220 min), or SOS (100 microM for 180 min) alone did not alter auditory neural thresholds. When infused together with glutamate, TBOA and SOS produced significant neural threshold shifts, leaving otoacoustic emissions intact. In addition, both TBOA and SOS exacerbated noise-induced hearing loss by producing larger neural threshold shifts and delaying recovery. DHK did not alter glutamate- or noise-induced hearing loss. The evidence points to a major role for GLAST, both in protecting the synapse from exposure to excess extracellular glutamate and in attenuating hearing loss due to acoustic overstimulation.

Fig. 1.

Infusion of glutamate (10 μM, 220 min), threo-beta-benzyloxyaspartate (TBOA; 200 μM, 220 min), or l-serine-O-sulfate (SOS 100 μM, 180 min) alone produced very little threshold shift (<5 dB) in compound action potentials (CAPs; A) or distortion product otoacoustic emissions (DPOAEs; B). A subsequent infusion of salicylate (5 mM, 10 min) served as a positive control, indicating drugs were getting to the organ of Corti. Salicylate produced 10–15 dB threshold shifts at high frequencies.

Fig. 2.

Infusion of glutamate (10 μM, 80 min) did not produce change in CAP (A) or DPOAE (B) thresholds. The infusion afterward of glutamate with TBOA or SOS resulted in 15–25 dB CAP threshold shifts at high frequencies with smaller shifts in low frequencies, leaving DPOAE intact. Infusion with the glutamate transporter-1 (GLT1) blocker, dihydrokainate (DHK), with 10 μM glutamate had no effect on either CAP or DPOAE thresholds.

Fig. 3.

Mice were exposed to a pure tone (22.3 kHz, 102 dB for 10 min) and CAPs recorded at 32 kHz and plotted (means ± SE). With infusion only of artificial perilymph (AP), CAP thresholds were shifted 23 dB at ∼30 s after termination of the noise exposure. (Data following noise exposure during infusion of artificial perilymph is replotted in each of the 3 panels.) Thresholds recovered gradually and returned to preexposure levels at ∼10 min. The infusion of TBOA (200 μM; A) or SOS (100 μM; B) during the noise exposure produced ∼33 dB CAP threshold shift at ∼30 s after noise. The CAP thresholds recovered ∼15 dB in the 1st 300 s after noise and then recovered more gradually to eventually reach near the preexposure level. The infusion of DHK (1 mM, GLT1 specific inhibitor; C) did not produce much change in the noise-induced reversible CAP threshold shifts. To quantify the changes associated with drug infusion, regression lines were fit to the data during the 1st 500 s following exposure, and slopes (recovery rates) and intercepts (initial shift) determined for each mouse (n = 6 each for AP, SOS, and TBOA; n = 4 for DHK). Recovery rates over the 1st 500 s were similar in all conditions. Changes in intercepts (initial shift) were significantly higher (P < .0025, Student's t-test) with TBOA and SOS compared with AP. Changes in intercepts with DHK were not significantly different from AP (P = 0.6).