4. Results
4.2 c-Fos Expression Across Cortical Areas
4.2.1 c-Fos Expression Across Cortical Areas in Sham and Control Animals
To determine if there was a difference in c-Fos expression between shams and controls, a mixed repeated measures ANOVA was conducted. c-Fos labeled cells in the auditory, multisensory and visual areas were averaged across all coronal sections (bregma 4.5, -5.0, -5.5, -6.0 and -6.5) to achieve a total c-Fos expression in each area [Figure 4.3].
There was a significant main effect of area (F(2,16)=11.643, p=0.001) but no interaction between area and condition (F(2,16)=1.832, p=0.192). Post-hoc tests using a Bonferroni correction revealed a significant difference between c-Fos expression in the auditory and multisensory cortices (p=0.025) and the auditory and visual cortices (p=0.008), but no difference between the multisensory and visual cortices (p=0.974). More importantly, there was no significant difference in c-Fos expression between sham and control animals (p=0.224). Therefore, for the remaining discussion control animals will be disregarded.
Figure 4.3: c-Fos expression across all coronal sections (bregma -4.5 to -6.5) in the auditory, multisensory and visual cortices of sham and control animals. A mixed repeated measures ANOVA revealed a significant main effect of area between sham and control animals (p=0.001). Post-hoc analysis revealed no difference between sham and control animals or noise-exposed and sham animals in any area. Error bars represent standard error of the mean.
4.2.2 c-Fos Expression at Varying Distances from Bregma
c-Fos expression was analyzed between sham and noise-exposed animals at varying distances from bregma in the auditory, multisensory and visual cortices individually.
Using a mixed repeated measures ANOVA in the auditory cortex (Figure 4.4 A), there was no significant main effect of distance from bregma (F(4,32)=1.575, p=0.205) and no interaction between distance from bregma and condition (F(4,32)=1.276, p=0.300).
Similarly in the multisensory cortex (Figure 4.4 B), there was no main effect of distance from bregma (F(4,32)=1.088, p=0.379) or interaction between distance from bregma and condition (F(4,32)=0.456, p=0.767). Lastly in the visual cortex (Figure 4.4 C), there was no significant main effect of distance from bregma (F(4,32)=1.363, p=0.269), or
interaction between distance from bregma and condition (F(4,32)=0.355, p=0.839). In conclusion, there was no significant difference in c-Fos expression at varying distances from bregma, therefore it is possible to collapse across distances from bregma and compare overall c-Fos expression between the auditory, multisensory and visual cortices in both sham and noise-exposed animals.
Figure 4.4 : c-Fos expression in the auditory (Panel A), multisensory (Panel B) and visual (Panel C) cortices at varying distances from bregma (-4.5, -5.0 -5.5, -6.0 and -6.5).
Error bars represent standard error of the mean.
4.2.3 c-Fos Expression Across Cortical Areas of Sham and Noise-Exposed Animals
c-Fos labeled cells in the auditory, multisensory and visual areas were averaged across all coronal sections (bregma -4.5, -5.0, -5.5, -6.0 and -6.5) to achieve a total c-Fos
expression in each area for noise-exposed and sham animals [Figure 4.5]. Using a mixed repeated measures ANOVA, there was a significant main effect of area (F(2,16)=14.409, p<0.001), but no interaction between area and condition (F(2,16)=8.892, p=0.090). Post-hoc analysis using a Bonferroni correction indicated a significant difference between c-Fos expression in the auditory and multisensory cortices (p=0.006) and the auditory and visual cortices (p=0.009), but no difference between the multisensory and visual cortices (p=0.232). Although there was no significant difference in c-Fos expression in sham and noise-exposed animals in any of the three areas, there is a trend where sham animals had consistently higher c-Fos expression levels compared to noise exposed animals.
Figure 4.5:c-Fos expression across all coronal sections (bregma -4.5 to -6.5) in the auditory, multisensory and visual cortices of sham and noise-exposed animals. A mixed repeated measures ANOVA revealed a significant main effect of area between sham and noise-exposed animals (p<0.001). Post-hoc analysis revealed no difference between noise-exposed and sham animals in any area. Error bars represent standard error of the mean.
Chapter 5
5. Discussion
The present study attempted to extend the findings of crossmodal plasticity in the auditory and multisensory cortices following hearing loss in adult animals. In 2009, Allman et al. reported significant crossmodal plasticity in adult ferrets, showing 84% of neurons in the primary auditory cortex responded to tactile stimulation following a profound hearing loss. In 2012, Meredith et al. subjected animals to a moderate hearing loss and also revealed a profound increase in the proportions of neurons in the auditory cortex responding to non-auditory cues (such as visual and tactile). Recent findings in our laboratory extended the findings to surrounding multisensory cortical areas,
demonstrating that after a moderate hearing impairment, animals have an increased visual responsiveness in the auditory and multisensory cortices compared to control animals.
The present experiment sought to evaluate whether c-Fos expression could serve as a complimentary technique to electrophysiological recordings recently shown in our laboratory and effectively map the increased responsiveness to visual stimuli that occurs in the auditory and multisensory cortices following noise exposure.
Adult Sprague-Dawley rats were subjected to an established noise-exposure procedure developed in the Allman laboratory. To confirm the noise-exposed animals had a significant hearing impairment, auditory brainstem response thresholds and wave I
amplitudes were measured before and fourteen days following the noise-exposure. Indeed, noise-exposed animals had a significant increase in threshold of 22±5 dB, and decrease in
and Liberman in 2009 demonstrated that even without an increase in hearing threshold, a decrease in wave I amplitude indicated significant damage to the auditory system. Thus both wave I amplitudes and thresholds were measured and confirmed the effectiveness of the established noise-exposure protocol.
c-Fos expression was assessed in the auditory, multisensory and visual cortices of noise-exposed, sham and control animals. Three groups were initially chosen because the sham and noise-exposed animals were retained in the animal facility for fourteen days during the experimental procedure. To rule out the possibility that animals contained in the animal facility were not exposed to certain confounding stimuli such as stress or noise that would affect the results, control animals were used. Control animals were taken from the animal facility, subjected to an initial ABR and visual stimulation procedure and immediately perfused. On the other hand, sham animals were given an initial ABR, maintained in the animal facility for fourteen days, subjected a final ABR then a visual stimulus and perfused. As expected, there was no difference in c-Fos expression between sham and noise-exposed animals in any area; thus, it was not necessary to analyze control animals farther and we concluded that there was no confounding variable in the animal facility that significantly altered the results.
When comparing sham and noise-exposed animals, there was a significant main effect of area, indicating that the visual stimulation appeared to have induced more c-Fos
expression in the visual and multisensory cortical areas compared to the primary auditory cortex. However, there was no difference between sham and noise-exposed animals in any area. Previous electrophysiological results in our laboratory indicated that animals subjected to a partial hearing loss had more visually responsive neurons in the primary
auditory and multisensory cortex compared to controls. Therefore, we expected to see an increase c-Fos labeled cells in noise-exposed animals compared to shams in the auditory and multisensory areas, and not in the visual area, which was not shown. It appears as if the visual stimulation was sufficient to evoke more c-Fos labeled cells in the visual and multisensory cortical areas compared to auditory areas in both animal groups, but not accurate enough to differentiate between noise-exposed and sham animals. Inconsistent with our previous electrophysiological studies, the molecular mapping of c-Fos was unable to demonstrate an increased responsiveness to visual stimulation that occurs in the auditory and multisensory cortices following noise exposure.
Compared to a previous study (Yamada et al., 1999), our visual stimulation protocol did not evoke sufficient levels of c-Fos to accurately differentiate between noise-exposed and sham animals, which may have been due to its short duration (6 min). Yamada et al.
found on average 100-150 c-Fos labeled cells over a cortical area of approximately 0.064 mm2, whereas the present study found on average 40-60 cells expressing c-Fos over a much larger area of approximately 0.85 mm2 [Figure 5.1]. A stimulation length of 6 minutes was chosen because pilot studies were focused on dual-labeling audiovisual cortical neurons using both c-Fos and another molecular marker, pERK. In order to effectively dual label, it was necessary to shorten the stimulation length for c-Fos. Thus future studies will use the same experimental procedure, except increase the visual stimulation time from 6 minutes to 45 minutes.
Figure 5.1: c-Fos expression in animals without a photic stimulation that were either naïve (panel A) or dark reared for one week (panel B), and animals subjected to a 45 minute photic stimulation after one week of dark rearing (panel C). Panel D shows c-Fos labeled cells in a 0.064 mm2 area in naïve (N) dark reared (D) and photic stimulated (PS) animals in several cortical layers. (Image from: Yamada et al. 1999, with permission).
Another possible explanation for the inconsistent results between molecular mapping of c-Fos and previous electrophysiological studies demonstrating an increased visual responsiveness in noise-exposed animals versus controls, is that molecular mapping may not be reliable in demonstrating cortical crossmodal plasticity. To our knowledge, no previous experiments have attempted to use this technique to study adult onset cortical plasticity following a partial hearing loss. Previously, it has been shown that basal expression of c-Fos is low, and it is highly up regulated after auditory (Zangenehpour &
Chaudhuri, 2001; Zuschratter et al, 1995) or visual (Zangenehpour & Chaudhuri, 2001;
Zangenehpour & Chaudhuri, 2002; Yamada et al, 1999) stimulation. Zuschratter et al in 1995 described increased c-Fos expression one hour after auditory tone burst stimulation in the primary auditory cortex in experimental animals compared to non-exposed controls.
Furthermore, Zangenehpour and Chaudhuri in 2001 demonstrated that expression of another immediate early gene, zif268, was elevated in the primary visual cortex and not in the primary auditory cortex following a visual stimulation. The present study is consistent with these results showing that c-Fos expression is in fact significantly
elevated in the visual cortex compared to the auditory cortex following visual stimulation.
Therefore, c-Fos expression might be sensitive enough to show increased expression in the core sensory areas following a stimulation procedure, but not to describe cortical crossmodal plasticity.
The discrepancies between molecular mapping and electrophysiology could also be explained due to counting c-Fos in all cortical layers. Previous experiments have described that early after sensory deprivation (approximately 10 days) cortical-cortical
However, 30 days following deprivation, there are changes to thalamocortical projections (Buonomano & Merzenich, 1998; Darian-Smith & Gilbert, 1995; Armstrong-James et al, 1994). Thus it would be more accurate to only analyze c-Fos labeled cells in cortical areas I-III where the majority of cortical-cortical fibers are located rather than all layers including those with thalamocortical projections.
In conclusion, molecular mapping of c-Fos was unable to demonstrate an increased responsiveness to visual stimulation that occurs in the auditory and multisensory cortices following noise exposure, which is inconsistent with our previous electrophysiological studies. Several explanations for the discrepancies are the short visual stimulation time, the sensitivity of molecular mapping and the cortical layers analyzed. Research in this field is necessary because it has previously been shown that when crossmodal plasticity in the auditory cortex is the most extensive, an individual is least likely to benefit from a cochlear implant (Lee et al, 2001). Also, the longer the delay to implantation, the less likely the cochlear implant will be successful (Proops et al 1999). More research must be conducted to assess the usefulness of molecular mapping as a tool for studying cortical crossmodal plasticity because it would allow a much wider area of the cortex to be analyzed compared to electrophysiology.
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