Auditory sensitivity regulation via rapid changes in expression of surface AMPA receptors

Abstract

We report a robust regulation of surface AMPA receptors in mouse auditory neurons, both with application of glutamate receptor agonists in cultured neurons and in response to acoustic stimulation in vivo. The reversible reduction of surface AMPA receptors following acoustic stimulation correlated with changes in acoustic sensitivity. Thus we show that AMPA receptor cycling is important for optimizing synaptic transfer at one of the most exacting synapses in the body.

Figure 1

Glutamate receptor agonists and antagonists altered surface and total GluR2 receptors in cultured mouse auditory neurons. (a) Differential interference contrast light microscopy, neurofilament label, and GluR2 label (total and surface) in auditory neurons. (b) Changes in surface and total GluR2 in response to application of 20 μM AMPA, NMDA or glutamate for 10 min. All glutamate agonists reversibly decreased surface GluR2 when compared with application of artificial perilymph (AP) (P < 0.05 for all times less than 40 min). Total GluR2 was reduced by NMDA and glutamate (P < 0.05), but not by AMPA application. Each data point represents the analysis of five images of neuronal clusters from each of three different cultures. The number of neurons in each image ranged from 3–40. Means ± s.e.m. are plotted. (c) Both NMDA and AMPA receptors mediated the removal of surface GluR2. Neither APV (50 μM) nor DNQX (20 μM) alone produced a significant (P < 0.05) reduction of surface GluR2. DNQX blocked AMPA-induced, but not NMDA-induced, removal of surface GluR2, whereas APV inhibited NMDA-induced, but not AMPA-induced, surface GluR2 removal. Plotted are means ± s.e.m. * P < 0.05, compared with control surface GluR2 as analyzed with a nonpaired Student’s t test.

Figure 2

Noise exposure reversibly decreased both cochlear surface AMPA receptor and auditory sensitivity in vivo. (a) Western blots of brain and cochlear extracts. The left two columns illustrate the total GluR2 in brain and cochlea. Surface GluR2 (columns 3–6) was determined after biotinylation of cochlear surface. (b) Following noise exposure, the time course of recovery of auditory sensitivity (reversal of threshold shift) correlated with that of the quantity of surface AMPA receptor. In ten mice, threshold shifts, measured after a 10-min broad-band noise exposure (1–40 kHz, 116 dB sound pressure level) were averaged over a broad range of cochlear frequencies (8, 20 and 45 kHz; threshold shifts for each of these frequencies are presented in Fig. 3c). For western blot analysis of surface GluR2, each data point represents five samples and each sample comprised cochlear tissue from three mice. Plotted are means ± s.e.m.

Figure 3

Pharmacology of surface GluR2 removal. (a,b) Myr-Dyn (200 μM for 90 min), APV + DNQX (3 mM each for 60 min) and APV (3 mM) alone all blocked noise-induced surface GluR2 removal. Each data point represents analysis of three samples and each sample comprised two cochleae (one cochlea taken per mouse). Data are expressed as mean ± s.e.m. and analyzed using a nonpaired Student’s t test. * P < 0.05, compared with cochlear surface GluR2 without noise. (c) APV reduced noise-induced ABR threshold shifts. The time course of recovery of threshold shifts for three different cochlear regions are shown in uninfused cochleae (noise only), cochleae infused with an artificial perilymph solution (AP + noise) and in those infused with APV (APV + noise). APV reduced the amount of threshold shift following acoustic overstimulation and reduced the time for recovery. Data with noise only were summarized from ten mice. Data for AP or APV perfusion were taken from 4–6 mice each. Means ± s.e.m. are plotted. * P < 0.05, comparing APV + noise with noise only.