Monaural conductive hearing loss alters the expression of the GluA3 AMPA and glycine receptor α1 subunits in bushy and fusiform cells of the cochlear nucleus

Abstract

The impact of conductive hearing loss (CHL), the second most common form of hearing loss, on neuronal plasticity in the central auditory pathway is unknown. After short-term (1 day) monaural earplugging, the GluA3 subunits of the AMPA receptor (AMPAR) are upregulated at auditory nerve synapses on the projection neurons of the cochlear nucleus; glycine receptor α1 (GlyRα1) subunits are downregulated at inhibitory synapses in the same neuronal population. These data suggest that CHL affects receptor trafficking at synapses. We examined the impact of 7 days of CHL on the general expression of excitatory and inhibitory receptors by quantitative biochemistry and immunohistochemistry, using specific antibodies to detect AMPAR subunits (GluA1, GluA2, GluA2/3, and GluA4), GlyRα1, and the GABA(A) receptor subunits β2/3. Following monaural earplugging and an elevation of the hearing threshold by approximately 35 dB, the immunolabeling of the antibody for the GluA2/3 subunits but not the GluA2 subunit increased on bushy cells (BCs) and fusiform cells (FCs) of the ipsilateral ventral and dorsal cochlear nuclei. These same cell types showed a downregulation of the GlyRα1 subunit. Similar results were observed in the contralateral nuclei. The expression levels of GABA(A) β2/3 were unchanged. These findings suggest that, following longer periods of monaural conductive hearing loss, the synthesis and subsequent composition of specific glutamate and glycine receptors in projection neurons and their synapses are altered; these changes may contribute to abnormal auditory processing.

Figure 1

Monaural earplugging effects on ABR thresholds. A) Representative ipsilateral ABR recordings before (top) and 20 min after earplugging (bottom). Sham control rats exhibited the characteristic ABR waveform at sound pressure levels 10 dB before and after the procedure. B) For monaural earplugged rats, ipsilateral ABR threshold was elevated about 40 dB 20 minutes after earplugging. C) Mean ABR thresholds are shown for clicks from ipsilateral and contralateral ears. Monaural plugged ears (ipsilateral) showed significant threshold elevation 20 mins after and one week following earplugging (P<0.05, 6 shams, 7 earplugged animals). Error bars represent SEMs.

Figure 2

Western blots of cochlear nucleus homogenates for GluA2/3 (A), GluA2 (B) and GlyRα1 (C) of normal hearing and plugged side. The bar on the right sight points out to the molecular weight of the proteins ~ 108 kD for GluA2/3 and GluA2 and ~ 48 kD for GlyRα1. Histograms show the gray value levels. (*P < 0.05).

Figure 3

Micrographs show immunoreaction for GluA2/3 in the AVCN of normal hearing (A) and monaural earplugged animals (B: ipsilateral plugged side; C: contralateral unplugged side). In A–C arrowheads = bushy cells. Scale bar : 50 µm. D, histogram of the RGVsoma values.

Figure 4

Micrographs show immunoreaction for GluA2/3 in the DCN of normal hearing (A) and monaural earplugged animals (B: ipsilateral plugged side; C: contralateral unplugged side). In A–C arrowheads = fusiform cells; arrows = cartwheel cells. ML: molecular layer; FCL: fusiform cell layer; DL: deep layer. Scale bar : 50 µm. D, histogram of the RGVsoma values.

Figure 5

Micrographs show immunoreaction for GluA2 in the DCN of normal hearing (A) and monaural earplugged animals (B: ipsilateral plugged side; C: contralateral unplugged side). In A–C arrowheads = fusiform cells; arrows = cartwheel cells. ML: molecular layer; FCL: fusiform cell layer; DL: deep layer. Scale bar : 50 µm. D, histogram of the RGVsoma values.

Figure 6

Micrographs show immunoreaction for GlyRα1 in the AVCN of normal hearing (A) and monaural earplugged animals (B: ipsilateral plugged side; C: contralateral unplugged side). In A–C arrowheads = bushy cells. Scale bar : 50 µm. D, histogram of the RGVsoma values.

Figure 7

Micrographs show immunoreaction for GlyRα1 in the DCN of normal hearing (A) and monaural earplugged animals (B: ipsilateral plugged side; C: contralateral unplugged side). In A–C arrowheads = putative fusiform cells; arrows = putative cartwheel cells. ML: molecular layer; FCL: fusiform cell layer; DL: deep layer. Scale bar: 50 µm. D, histogram of the RGVsoma values.

Figure 8

Micrographs show immunoreaction for GABA_A β2/3 in the VCN (A–B) and DCN (C–C’) of normal hearing animals. Scale bars AVCN: anteroventral cochlear nucleus; PVCN: posteroventral cochlear nucleus; DCN: dorsal cochlear nucleus.; D: dorsal; L: lateral Arrowheads: fusiform cells; arrow: multipolar cells. Scale bars, A–C: 200 µm; C’: 50 µm.