The GPG/Vall hamster is an animal model that exhibits seizures in response to sound stimulation. Since the superior colliculus (SC) is implicated in the neuronal network of audiogenic seizures (AGS) in other forms of AGS, this study evaluated seizure-related anatomical or neurochemical abnormalities in the SC of the GPG/Vall hamster. This involved calbindin (CB) and parvalbumin (PV) immunohistochemistry, densitometric analysis and high performance liquid chromatography in the superficial and deep layers of the SC in control and epileptic animals. Compared to control animals, a reduction in SC volume and a hypertrophy of neurons located in the deep layers of the SC were observed in the epileptic hamster. Although, analysis of CB-immunohistochemistry in the superficial layers did not show differences between groups, analysis of PV-immunostaining in the deep SC revealed an increase in the mean gray level within immunostained neurons as well as a decreased immunostained neuropil in the GPG/Vall hamster as compared to control animals. These alterations were accompanied by a decrease in the levels of GABA and increased levels of taurine in the epileptic animal. These data indicate that the deep SC of the GPG/Vall hamster is structurally abnormal; suggesting its involvement in the neuronal network for AGS.

Repeated noise exposure induces inflammation and cellular adaptations in the peripheral and central auditory system resulting in pathophysiology of hearing loss. In this study, we analyzed the mechanisms by which noise-induced inflammatory-related events in the cochlea activate glial-mediated cellular responses in the cochlear nucleus (CN), the first relay station of the auditory pathway. The auditory function, glial activation, modifications in gene expression and protein levels of inflammatory mediators and ultrastructural changes in glial-neuronal interactions were assessed in rats exposed to broadband noise (0.5-32 kHz, 118 dB SPL) for 4 h/day during 4 consecutive days to induce long-lasting hearing damage. Noise-exposed rats developed a permanent threshold shift which was associated with hair cell loss and reactive glia. Noise-induced microglial activation peaked in the cochlea between 1 and 10D post-lesion; their activation in the CN was more prolonged reaching maximum levels at 30D post-exposure. RT-PCR analyses of inflammatory-related genes expression in the cochlea demonstrated significant increases in the mRNA expression levels of pro- and anti-inflammatory cytokines, inducible nitric oxide synthase, intercellular adhesion molecule and tissue inhibitor of metalloproteinase-1 at 1 and 10D post-exposure. In noise-exposed cochleae, interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) were upregulated by reactive microglia, fibrocytes, and neurons at all time points examined. In the CN, however, neurons were the sole source of these cytokines. These observations suggest that noise exposure causes peripheral and central inflammatory reactions in which TNF-α and IL-1β are implicated in regulating the initiation and progression of noise-induced hearing loss.

We have previously reported that young adult rats exposed to daily, short-duration noise for extended time periods, develop accelerated presbycusis starting at 6 months of age. Auditory aging is associated with progressive hearing loss, cell deterioration, dysregulation of the antioxidant defense system, and chronic inflammation, among others. To further characterize cellular and molecular mechanisms at the crossroads between noise and age-related hearing loss (ARHL), 3-month-old rats were exposed to a noise-accelerated presbycusis (NAP) protocol and tested at 6 and 16 months of age, using auditory brainstem responses, Real-Time Reverse Transcription-Quantitative PCR (RT-qPCR) and immunocytochemistry. Chronic noise-exposure leading to permanent auditory threshold shifts in 6-month-old rats, resulted in impaired sodium/potassium activity, degenerative changes in the lateral wall and spiral ganglion, increased lipid peroxidation, and sustained cochlear inflammation with advancing age. Additionally, at 6 months, noise-exposed rats showed significant increases in the gene expression of antioxidant enzymes (superoxide dismutase 1/2, glutathione peroxidase 1, and catalase) and inflammation-associated molecules [ionized calcium binding adaptor molecule 1, interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha]. The levels of IL-1β were upregulated in the spiral ganglion and spiral ligament, particularly in type IV fibrocytes; these cells showed decreased levels of connective tissue growth factor and increased levels of 4-hydroxynonenal. These data provide functional, structural and molecular evidence that age-noise interaction contributes to exacerbating presbycusis in young rats by leading to progressive dysfunction and early degeneration of cochlear cells and structures. These findings contribute to a better understanding of NAP etiopathogenesis, which is essential as it affects the life quality of young adults worldwide.

Neurons of the cochlear nuclei receive axosomatic endings from primary afferent fibers from the cochlea and have projections that diverge to form parallel ascending auditory pathways. These cells are characterized by neurochemical phenotypes such as levels of calretinin. To test whether or not early deafferentation results in changes in calretinin immunostaining in the cochlear nucleus, unilateral cochlear ablations were performed in ferrets soon after hearing onset (postnatal day [P]30-P40). Two months later, changes in calretinin immunostaining as well as cell size, volume, and synaptophysin immunostaining were assessed in the anteroventral (AVCN), posteroventral (PVCN), and dorsal cochlear nucleus (DCN). A decrease in calretinin immunostaining was evident ipsilaterally within the AVCN and PVCN but not in the DCN. Further analysis revealed a decrease both in the calretinin-immunostained neuropil and in the calretinin-immunostained area within AVCN and PVCN neurons. These declines were accompanied by significant ipsilateral decreases in volume as well as neuron area in the AVCN and PVCN compared with the contralateral cochlear nucleus and unoperated animals, but not compared with the DCN. In addition, there was a significant contralateral increase in calretinin-immunostained area within AVCN and PVCN neurons compared with control animals. Finally, a decrease in area of synaptophysin immunostaining in both the ipsilateral AVCN and PVCN without changes in the number of boutons was found. The present data demonstrate that unilateral cochlear ablation leads to 1) decreased immunostaining of the neuropil in the AVCN and PVCN ipsilaterally, 2) decreased calretinin immunostaining within AVCN and PVCN neurons ipsilaterally, 3) synaptogenesis in the AVCN and PVCN ipsilaterally, and 4) increased calretinin immunostaining within AVCN and PVCN neurons contralaterally.

The removal of afferent activity has been reported to modify neuronal activity in the cochlear nucleus of adult rats. After cell damage, microglial cells are rapidly activated, initiating a series of cellular responses that influences neuronal function and survival. To investigate how this glial response occurs and how it might influence injured neurons, bilateral cochlear ablations were performed on adult rats to examine the short-term (16 and 24 hours and 4 and 7 days) and long-term (15, 30, and 100 days) changes in the distribution and morphology of microglial cells (immunostained with the ionized calcium-binding adaptor molecule 1; Iba-1) and the interaction of microglial cells with deafferented neurons in the ventral cochlear nucleus. A significant increase in the mean cross-sectional area and Iba-1 immunostaining of microglial cells in the cochlear nucleus was observed at all survival times after the ablation compared with control animals. These increases were concomitant with an increase in the area of Iba-1 immunostaining at 24 hours and 4, 7, and 15 days postablation. Additionally, microglial cells were frequently seen apposing the cell bodies and dendrites of auditory neurons at 7, 15, and 30 days postablation. In summary, these results provide evidence for persistent glial activation in the ventral cochlear nucleus and suggest that long-term interaction occurs between microglial cells and deafferented cochlear nucleus neurons following bilateral cochlear ablation, which could facilitate the remodeling of the affected neuronal circuits.

The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) requires adequate normalization in order to ensure accurate results. The use of reference genes is the most common method to normalize RT-qPCR assays; however, many studies have reported that the expression of frequently used reference genes is more variable than expected, depending on experimental conditions. Consequently, proper validation of the stability of reference genes is an essential step when performing new gene expression studies. Despite the fact that RT-qPCR has been widely used to elucidate molecular correlates of noise-induced hearing loss (NIHL), up to date there are no reports demonstrating validation of reference genes for the evaluation of changes in gene expression after NIHL. Therefore, in this study we evaluated the expression of some commonly used reference genes (Arbp, b-Act, b2m, CyA, Gapdh, Hprt1, Tbp, Tfrc and UbC) and examined their suitability as endogenous control genes for RT-qPCR analysis in the adult Wistar rat in response to NIHL. Four groups of rats were noise-exposed to generate permanent cochlear damage. Cochleae were collected at different time points after noise exposure and the expression level of candidate reference genes was evaluated by RT-qPCR using geNorm, NormFinder and BestKeeper software to determine expression stability. The three independent applications revealed Tbp as the most stably expressed reference gene. We also suggest a group of top-ranked reference genes that can be combined to obtain suitable reference gene pairs for the evaluation of the effects of noise on gene expression in the cochlea. These findings provide essential basis for further RT-qPCR analysis in studies of NIHL using Wistar rats as animal model.

Both age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL) may share pathophysiological mechanisms in that they are associated with excess free radical formation and cochlear blood flow reduction, leading to cochlear damage. Therefore, it is possible that short, but repeated exposures to relatively loud noise during extended time periods, like in leisure (i.e., musical devices and concerts) or occupational noise exposures, may add to cochlear aging mechanisms, having an impact on the onset and/or progression of ARHL. Consequently, the aim of the present study was to determine if repeated short-duration overexposure to a long-term noise could accelerate permanent auditory threshold shifts associated with auditory aging in an animal model of ARHL. Toward this goal, young adult, 3-month-old Wistar rats were divided into two groups: one exposed (E) and the other non-exposed (NE) to noise overstimulation. The stimulation protocol consisted of 1 h continuous white noise at 110 dB sound pressure level (SPL), 5 days a week, allowing 2 days for threshold recovery before initiating another stimulation round, until the animals reached an age of 18 months. Auditory brainstem response (ABR) recordings at 0.5, 1, 2, 4, 8, 16, and 32 kHz were performed at 3, 6, 12, and 18 months of age. The results demonstrate that in the E group there were significant increases in auditory thresholds at all tested frequencies starting already at 6 months of age, which extended at 12 and 18 months. However, in NE animals threshold shifts were not evident until 12 months, extending to 18 months of age. Threshold shifts observed in the E animals at 6 and 12 months were significantly larger than those observed in the NE group at the same ages. Threshold shifts at 6 and 12 months in E animals resembled those at 12 and 18 months in NE animals, respectively. This suggests that repeated noise overstimulation in short-duration episodes accelerates the time-course of hearing loss in this animal model of ARHL.

One of the main mechanisms used by neurons and glial cells to promote repair following brain injury is to upregulate activity-dependent molecules such as insulin-like growth factor 1 (IGF-1) and interleukin-1β (IL-1β). In the auditory system, IGF-1 is crucial for restoring synaptic transmission following hearing loss; however, whether IL-1β is also involved in this process is unknown. In this study, we evaluated the expression of IGF-1 and IL-1β within neurons and glial cells of the ventral cochlear nucleus in adult rats at 1, 7, 15, and 30 days following bilateral cochlear ablation. After the lesion, significant increases in both the overall mean gray levels of IGF-1 immunostaining and the mean gray levels within cells of the cochlear nucleus were observed at 1, 7, and 15 days compared with control animals. The expression and distribution of IL-1β in the ventral cochlear nucleus of ablated animals was temporally and spatially correlated with IGF-1. We also observed a lack of colocalization between IGF-1 and IL-1β with either astrocytes or microglia at any of the time points following ablation. These results suggest that the upregulation of IGF-1 and IL-1β levels within neurons-but not within glial cells-may reflect a plastic mechanism involved in repairing synaptic homeostasis of the overall cellular environment of the cochlear nucleus following bilateral cochlear ablation.

Calretinin (CR) is a calcium-binding protein that plays an important role in the homeostasis of intracellular calcium concentration in the auditory pathway. To test if hearing loss could lead indirectly to modifications in levels of this calcium-binding protein in neurons and neuropilar structures outside of the lemniscal auditory pathway, CR-immunostaining was evaluated in the superior colliculus (SC) in adult ferrets at 1, 20 and 90 days after unilateral cochlear ablation. The results demonstrate that within 24h there was a significant increase in CR-immunostaining in ablated animals as indicated by an increase in the mean gray level of immunostaining in the deep, multisensory layers of the contralateral SC compared to the ipsilateral side and control ferrets. This upregulation was evident in both neurons and neuropil and did not change at the two subsequent time points. In contrast, there was no change in the superficial layers of the SC which have visual properties but no auditory inputs. These findings suggest that upregulation of CR levels within neurons and neuropil in the contralateral deep SC is subject to modifications by activity in multisynaptic auditory pathways. Therefore, cochlear-driven activity appears to affect calcium-binding protein levels not only in auditory nuclei but also in other neural structures whose response properties may be influenced by auditory-related activity.

In the central nucleus of the inferior colliculus (IC), afferent projections are aligned with dendritic arbors of disk-shaped cells, forming fibrodendritic layers. One feature that may serve as a guide for study of the intrinsic organization of the IC layers is the segregation of certain inputs to bands and patches within the layers of the central nucleus. In this study, we used Phaseolus leucoagglutinin as an anterograde tracer to examine the projections from the dorsal nucleus of the lateral lemniscus to the contralateral IC in adult ferrets. The labeled afferent projections distributed along the IC layers in a series of bands where there were dense endings and interband spaces where there were few if any endings. Branches of individual labeled axons that were reconstructed distributed within a single afferent band. Measurements of both the terminal density distribution and the optical density across the band were similar indicating that afferent bands were approximately 85 microm thick. Quantitative measurements of the labeled afferent bands will enhance comparison with other afferent projections and analysis of afferent development and plasticity.