Diminished cortical inhibition in an aging mouse model of chronic tinnitus

Abstract

Flavoprotein autofluorescence imaging was used to examine auditory cortical synaptic responses in aged animals with behavioral evidence of tinnitus and hearing loss. Mice were exposed to noise trauma at 1-3 months of age and were assessed for behavioral evidence of tinnitus and hearing loss immediately after the noise trauma and again at ~24-30 months of age. Within 2 months of the final behavioral assessment, auditory cortical synaptic transmission was examined in brain slices using electrical stimulation of putative thalamocortical afferents, and flavoprotein autofluorescence imaging was used to measure cortical activation. Noise-exposed animals showed a 68% increase in amplitude of cortical activation compared with controls (p = 0.008), and these animals showed a diminished sensitivity to GABA(A)ergic blockade (p = 0.008, using bath-applied 200 nm SR 95531 [6-Imino-3-(4-methoxyphenyl)-1(6H)-p yridazinebutanoic acid hydrobromide]). The strength of cortical activation was significantly correlated to the degree of tinnitus behavior, assessed via a loss of gap detection in a startle paradigm. The decrease in GABA(A) sensitivity was greater in the regions of the cortex farther away from the stimulation site, potentially reflecting a greater sensitivity of corticocortical versus thalamocortical projections to the effects of noise trauma. Finally, there was no relationship between auditory cortical activation and activation of the somatosensory cortex in the same slices, suggesting that the increases in auditory cortical activation were not attributable to a generalized hyperexcitable state in noise-exposed animals. These data suggest that noise trauma can cause long-lasting changes in the auditory cortical physiology and may provide specific targets to ameliorate the effects of chronic tinnitus.

Figures

Figure 1.

Behavioral assessment of hearing loss and tinnitus behavior in noise-exposed animals. The top images illustrate temporal sequences of sounds used to assess PPI (A) and gap detection (B). Bar graphs on the left illustrate the mean and SE of the PPI ratio (defined in Materials and Methods) of control animals (n = 4) and noise-exposed animals (n = 6). Bar graphs on the right illustrate mean and SE values for the gap detection ratio of control animals (n = 4) and noise-exposed animals (n = 6). p values are derived from Mann–Whitney U test.

Figure 2.

A–D, Δf/f maps of auditory cortical activations superimposed on raw fluorescence images under conditions of 100 μA stimulus and normal ACSF (A), 300 μA stimulus and normal ACSF (B), 100 μA stimulus and 100 nm SR 95331 (C), and 300 μA stimulus and 200 nm SR 95531 (D). Small dotted arrows show the stimulus site. R, Rostral; C, caudal; L, lateral; M, medial. Color bar shows percentage change in fluorescence relative to baseline. E, Percentage change in fluorescence over time (averaged over 5 runs, presented 20 s apart), before any drugs were presented (Control), after exposure to 100 μm APV and 80 μm DNQX (APV + DNQX), after washout of APV and DNQX (Wash), or after exposure to 1 μm tetrodotoxin (TTX). F, Amplitude, expressed as maximum percentage increase in fluorescence, at a series of stimulus amplitudes from 25 to 500 μA, for slice shown in A–D. Raw data were fit with a four-parameter sigmoid model. Nissl staining (G) and parvalbumin immunostaining (H) of sections taken from the same slice.

Figure 3.

Examples of Δf/f cortical activation maps for a control animal (A) and a noise-exposed animal (B). Stimulus amplitudes/parameters were identical: 300 μA, 2 ms duration pulses at 40 pulses/s for 1 s. Color bar corresponds to the percentage increase in fluorescence over baseline.

Figure 4.

Comparison of strength of cortical activation and GABAAergic sensitivity in the on-beam and off-beam regions of the auditory cortex (left and right, respectively). Top row shows the maximum increase in fluorescence. Bottom row shows the ratio of the response under conditions of exposure to 200 nm SR 95331 to normal ACSF. Each diamond or triangle represents a single slice. Horizontal lines represent mean value. P values are derived from Mann–Whitney U test.

Figure 5.

Comparison of strength of cortical activation in the infragranular and supragranular layers of the auditory cortex (A and B, respectively). Each diamond or triangle represents a single slice. Horizontal lines represent mean value. P values are derived from Mann–Whitney U test.

Figure 6.

A, Amplitude of the on-beam cortical response in the animals that were tested for APV sensitivity. B, Amplitude of the cortical activation in the same slices shown in A after exposure to 100 μm APV. C, The ratio of the cortical response under conditions of APV exposure to conditions of normal ACSF exposure. Each diamond or triangle represents a single slice. Horizontal lines represent mean value. p values are derived from Mann–Whitney U test.

Figure 7.

A, Diagram illustrating the relative locations of stimulating electrodes for auditory cortex (AC) stimulation (left) and secondary somatosensory cortex (S2) stimulation (right). B, Cytochrome oxidase-stained section from same slice, demonstrating lack of barrel architecture in the region of cortical activation. SCWM, Subcortical white matter; R, rostral; C, caudal; L, lateral; M, medial. C, Example of a response in secondary somatosensory cortex to 200 μA stimulus to the subcortical white matter. D, Correlation between the magnitude of activation in the auditory cortex to the magnitude of activation in the somatosensory cortex in the same slices. Black circles, Noise-exposed animals; black triangles, sham-treated animals. R2 and p values generated from Spearman's correlations.

Figure 8.

A, Correlation between maximum increase in fluorescence versus gap startle ratio at 12.5 kHz. B, Correlation between maximum increase in fluorescence versus PPI ratio at 16 kHz. Black diamonds, Noise-exposed animals; black triangles, sham-treated animals. R2 and p values generated from Spearman's correlations.