Candidate Key Proteins in Tinnitus: A Bioinformatic Study of Synaptic Transmission in Spiral Ganglion Neurons.

To study key proteins associated with changes in synaptic transmission in the spiral ganglion in tinnitus, we build three gene lists from the GeneCard database: 1. Perception of sound (PoS), 2. Acoustic stimulation (AcouStim), and 3. Tinnitus (Tin). Enrichment analysis by the DAVID database resulted in similar Gene Ontology (GO) terms for cellular components in all gene lists, reflecting synaptic structures known to be involved in auditory processing. The STRING protein-protein interaction (PPI) network and the Cytoscape data analyzer were used to identify the top two high-degree proteins (HDPs) and their high-score interaction proteins (HSIPs) identified by the combined score (CS) of the corresponding edges. The top two protein pairs (key proteins) for the PoS are BDNF-GDNF and OTOF-CACNA1D and for the AcouStim process BDNF-NTRK2 and TH-CALB1. The Tin process showed BDNF and NGF as HDPs, with high-score interactions with NTRK1 and NGFR at a comparable level. Compared to the PoS and AcouStim process, the number of HSIPs of key proteins (CS > 90. percentile) increases strongly in Tin. In the PoS and AcouStim networks, BDNF receptor signaling is the dominant pathway, and in the Tin network, the NGF-signaling pathway is of similar importance. Key proteins and their HSIPs are good indicators of biological processes and of signaling pathways characteristic for the normal hearing on the one hand and tinnitus on the other.

Therapeutic Approaches to the Treatment of Tinnitus.

Tinnitus is a highly prevalent condition that is associated with hearing loss in most cases. In the absence of external stimuli, phantom perceptions of sounds emerge from alterations in neuronal activity within central auditory and nonauditory structures. Pioneering studies using lidocaine revealed that tinnitus is susceptible to pharmacological interventions. However, lidocaine is not effective in all patients, and no other drug has been identified with clear efficacy for the long-term treatment of tinnitus. In this review, we present recent advances in tinnitus research, including more detailed knowledge of its pathophysiology and involved neurotransmitter systems. Moreover, we summarize results from animal and clinical treatment studies as well as from studies that identified tinnitus as a side effect of pharmacological treatments. Finally, we focus on challenges in the development of pharmacological compounds for the treatment of tinnitus, namely the limitations of available animal models and of standardized clinical research methodologies.

Interactions between the hippocampus and the auditory pathway.

Increasing evidence has shown that noise overexposure could lead to impaired hippocampal function. Hippocampal alteration is also observed in several auditory deficits, including hearing loss, and tinnitus. Therefore, the functions of hearing and cognition interact with each other. Here, we summarize the evidence that noise affects the hippocampus from aspects of behavior, neurogenesis, ultrastructure, neurotransmission, other biomarkers, and electrophysiology. We also address hippocampal alterations in auditory disorders, including hearing loss and tinnitus. Based on the current state of the field, we point out several aspects that need further investigation. This review is not only to provide a comprehensive summary of the current state of the field but to emphasize that hearing matters in cognition and pave the way for future research.

Noise Exposure Alters Glutamatergic and GABAergic Synaptic Connectivity in the Hippocampus and Its Relevance to Tinnitus.

Accumulating evidence implicates a role for brain structures outside the ascending auditory pathway in tinnitus, the phantom perception of sound. In addition to other factors such as age-dependent hearing loss, high-level sound exposure is a prominent cause of tinnitus. Here, we examined how noise exposure altered the distribution of excitatory and inhibitory synaptic inputs in the guinea pig hippocampus and determined whether these changes were associated with tinnitus. In experiment one, guinea pigs were overexposed to unilateral narrow-band noise (98 dB SPL, 2 h). Two weeks later, the density of excitatory (VGLUT-1/2) and inhibitory (VGAT) synaptic terminals in CA1, CA3, and dentate gyrus hippocampal subregions was assessed by immunohistochemistry. Overall, VGLUT-1 density primarily increased, while VGAT density decreased significantly in many regions. Then, to assess whether the noise-induced alterations were persistent and related to tinnitus, experiment two utilized a noise-exposure paradigm shown to induce tinnitus and assessed tinnitus development which was assessed using gap-prepulse inhibition of the acoustic startle (GPIAS). Twelve weeks after sound overexposure, changes in excitatory synaptic terminal density had largely recovered regardless of tinnitus status, but the recovery of GABAergic terminal density was dramatically different in animals expressing tinnitus relative to animals resistant to tinnitus. In resistant animals, inhibitory synapse density recovered to preexposure levels, but in animals expressing tinnitus, inhibitory synapse density remained chronically diminished. Taken together, our results suggest that noise exposure induces striking changes in the balance of excitatory and inhibitory synaptic inputs throughout the hippocampus and reveal a potential role for rebounding inhibition in the hippocampus as a protective factor leading to tinnitus resilience.

Neuroprotective Effect of Valproic Acid on Salicylate-Induced Tinnitus.

High-dose salicylate induces temporary moderate hearing loss and the perception of a high-pitched tinnitus in humans and animals. Previous studies demonstrated that high doses of salicylate increase N-methyl-d-aspartate (NMDA) receptor levels, resulting in a rise in Ca2+ influx and induction of excitotoxicity. Glutamate excitotoxicity is associated with failure in the maintenance of calcium homeostasis, mitochondrial dysfunction, and production of reactive oxygen species (ROS). Valproic acid (VPA) is widely used for the management of bipolar disorder, epilepsy, and migraine headaches, and is known to regulate NMDA receptor activity. In this study, we examined the beneficial effects of VPA in a salicylate-induced tinnitus model in vitro and in vivo. Cells were pretreated with VPA followed by salicylate treatment. The expression levels of NMDA receptor subunit NR2B, phosphorylated cAMP response element-binding protein-an apoptosis marker, and intracellular levels of ROS were measured using several biochemical techniques. We observed increased expression of NR2B and its related genes TNFα and ARC, increased intracellular ROS levels, and induced expression of cleaved caspase-3. These salicylate-induced changes were attenuated in the neuronal cell line SH-SY5Y and rat cortical neurons after VPA pretreatment. Together, these results provide evidence of the beneficial effects of VPA in a salicylate-induced temporary hearing loss and tinnitus model.

Tinnitus Correlates with Downregulation of Cortical Glutamate Decarboxylase 65 Expression But Not Auditory Cortical Map Reorganization.

Hearing loss is the biggest risk factor for tinnitus, and hearing-loss-related pathological changes in the auditory pathway have been hypothesized as the mechanism underlying tinnitus. However, due to the comorbidity of tinnitus and hearing loss, it has been difficult to differentiate between neural correlates of tinnitus and consequences of hearing loss. In this study, we dissociated tinnitus and hearing loss in FVB mice, which exhibit robust resistance to tinnitus following monaural noise-induced hearing loss. Furthermore, knock-down of glutamate decarboxylase 65 (GAD65) expression in auditory cortex (AI) by RNA interference gave rise to tinnitus in normal-hearing FVB mice. We found that tinnitus was significantly correlated with downregulation of GAD65 in the AI. By contrast, cortical map distortions, which have been hypothesized as a mechanism underlying tinnitus, were correlated with hearing loss but not tinnitus. Our findings suggest new strategies for the rehabilitation of tinnitus and other phantom sensation, such as phantom pain.SIGNIFICANCE STATEMENT Hearing loss is the biggest risk factor for tinnitus in humans. Most animal models of tinnitus also exhibit comorbid hearing loss, making it difficult to dissociate the mechanisms underlying tinnitus from mere consequences of hearing loss. Here we show that, although both C57BL/6 and FVB mice exhibited similar noise-induced hearing threshold increase, only C57BL/6, but not FVB, mice developed tinnitus following noise exposure. Although both strains showed frequency map reorganization following noise-induced hearing loss, only C57BL/6 mice had reduced glutamate decarboxylase 65 (GAD65) expression in the auditory cortex (AI). Knocking down GAD65 expression in the AI resulted in tinnitus in normal-hearing FVB mice. Our results suggest that reduced inhibitory neuronal function, but not sensory map reorganization, underlies noise-induced tinnitus.

Aberrant expression of Nav1.6 in the cochlear nucleus correlates with salicylate-induced tinnitus in rats.

Hyperactivity in cochlear nucleus (CN) is one of the major neural correlates for tinnitus induction, yet the molecular factors that participate in the neuronal hyperexcitability remain unclear. The present study showed that acute and chronic administrations of salicylate were both capable of inducing reversible tinnitus in rats. The number of GAD 65/67-immunoreactive neurons in the AVCN and DCN was decreased, while the number of VGLUT 1/2-immunoreactive neurons in the AVCN and DCN was increased when rats were experiencing tinnitus, providing evidence for excitatory-inhibitory imbalance in CN is correlated with tinnitus. Interestingly, the expression level of Nav1.6, an important subtype of voltage-gated sodium channels was significantly increased in the DCN and AVCN of rats experiencing tinnitus, the up-regulation of Nav1.6 was returned to normal level following the disappearance of tinnitus. Double-labeling experiments revealed that Nav1.6 expression was down-regulated in the GAD 65/67-positive neurons in the DCN and AVCN of rats experiencing tinnitus. Notably, the percentage of co-localization of Nav1.6 and NeuN-labeling fusiform neurons was markedly increased in the DCN during tinnitus. These findings uncover the tinnitus-associated alteration in Nav1.6, a potential key contributor that can lead to hyperexcitability in CN and contribute to salicylate-induced tinnitus.

Remodeling of cholinergic input to the hippocampus after noise exposure and tinnitus induction in Guinea pigs.

Here, we investigate remodeling of hippocampal cholinergic inputs after noise exposure and determine the relevance of these changes to tinnitus. To assess the effects of noise exposure on the hippocampus, guinea pigs were exposed to unilateral noise for 2 hr and 2 weeks later, immunohistochemistry was performed on hippocampal sections to examine vesicular acetylcholine transporter (VAChT) expression. To evaluate whether the changes in VAChT were relevant to tinnitus, another group of animals was exposed to the same noise band twice to induce tinnitus, which was assessed using gap-prepulse Inhibition of the acoustic startle (GPIAS) 12 weeks after the first noise exposure, followed by immunohistochemistry. Acoustic Brainstem Response (ABR) thresholds were elevated immediately after noise exposure for all experimental animals but returned to baseline levels several days after noise exposure. ABR wave I amplitude-intensity functions did not show any changes after 2 or 12 weeks of recovery compared to baseline levels. In animals assessed 2-weeks following noise-exposure, hippocampal VAChT puncta density decreased on both sides of the brain by 20-60% in exposed animals. By 12 weeks following the initial noise exposure, changes in VAChT puncta density largely recovered to baseline levels in exposed animals that did not develop tinnitus, but remained diminished in animals that developed tinnitus. These tinnitus-specific changes were particularly prominent in hippocampal synapse-rich layers of the dentate gyrus and areas CA3 and CA1, and VAChT density in these regions negatively correlated with tinnitus severity. The robust changes in VAChT labeling in the hippocampus 2 weeks after noise exposure suggest involvement of this circuitry in auditory processing. After chronic tinnitus induction, tinnitus-specific changes occurred in synapse-rich layers of the hippocampus, suggesting that synaptic processing in the hippocampus may play an important role in the pathophysiology of tinnitus.

Chronic tinnitus and BDNF/GDNF CpG promoter methylations: a case-control study.

Brain-derived neurotrophic factor (BDNF) and Glial-derived neurotrophic factor (GDNF) are neurotrophic factors that play key roles in the auditory pathway. While the relationship between serum levels and polymorphisms of BDNF/GDNF and chronic tinnitus is emphasized in the literature, there is no study showing the link between the promoter methylations of these genes and tinnitus. For this purpose, the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations was investigated to identify their role in the pathophysiology of tinnitus. In this case-control study, we examined the possible effects of BDNF/GDNF methylations in the blood samples of patients with tinnitus complaints for more than 3 months. Sixty tinnitus subjects between the ages of 18-55 and 50 healthy control subjects in the same age group who were free of any otorhinolaryngology and systemic disease were selected for examination. Methylation of total 12 CpG sites in BDNF and GDNF promoter regions were determined by the bisulfite-pyrosequencing method. Statistically significant differences were detected between BDNF CpG6 and GDNF CpG3-5-6 methylation ratios in the comparison of control group and the chronic tinnitus patients (P = 0.002, 0.0005, 0.00003, and 0.0029, respectively). To our knowledge, this is the first study in the literature investigating the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations. It is believed that the current results might be supported by investigating the relationships between BDNF/GDNF methylations and genotypes in future research using higher sample sizes.

Blast-induced tinnitus: Animal models.

Blast-induced tinnitus is a prevalent problem among military personnel and veterans, as blast-related trauma damages the vulnerable microstructures within the cochlea, impacts auditory and non-auditory brain structures, and causes tinnitus and other disorders. Thus far, there is no effective treatment of blast-induced tinnitus due to an incomplete understanding of its underlying mechanisms, necessitating development of reliable animal models. This article focuses on recent animal studies using behavioral, electrophysiological, imaging, and pharmacological tools. The mechanisms underlying blast-induced tinnitus are largely similar to those underlying noise-induced tinnitus: increased spontaneous firing rates, bursting, and neurosynchrony, Mn++ accumulation, and elevated excitatory synaptic transmission. The differences mainly lie in the data variability and time course. Noise trauma-induced tinnitus mainly originates from direct peripheral deafferentation at the cochlea, and its etiology subsequently develops along the ascending auditory pathways. Blast trauma-induced tinnitus, on the other hand, results from simultaneous impact on both the peripheral and central auditory systems, and the resultant maladaptive neuroplasticity may also be related to the additional traumatic brain injury. Consequently, the neural correlates of blast-induced tinnitus have different time courses and less uniform manifestations of its neural correlates.

Up-regulation of Ca2+/CaMKII/CREB signaling in salicylate-induced tinnitus in rats.

The purpose of the study was to investigate the changes of Ca2+/calmodulin-dependent protein kinases II (CaMKII)/cAMP response element-binding protein (CREB) signaling pathway in a rat tinnitus model. Eighteen Wistar rats were randomly divided into three groups: normal control (NC), normal saline (NS), and tinnitus model (TM) groups. Tinnitus model was induced by intraperitoneal injection of salicylate. The concentration of intracellular calcium level in auditory cortex cells was determined using Fura-2 acetoxymethyl ester (Fura-2 AM) method with fluorospectrophotometer. Expressions of calmodulin (CaM), N-methyl-D-aspartate receptor 2B subunit (NR2B), calcium-calmodulin kinase II (CaMKII), and cAMP response element-binding protein (CREB) were detected with Western blot. Tinnitus model was successfully established by the intraperitoneal administration of salicylate in rats. Compared with rats in NC and NS groups, salicylate administration significantly elevated CaM, NR2B, phospho-CaMKII and phospho-CREB expression in auditory cortex from tinnitus model group (p < 0.05), and the free intracellular Ca2+ concentrations (p < 0.05). Our data reveal that salicylate administration causes tinnitus symptoms and elevates Ca2+/CaMKII/CREB signaling pathway in auditory cortex cells. Our study likely provides a new understanding of the development of tinnitus.

Effects of Spirulina on GABA Receptor Gene Expression in Salicylate-Induced Tinnitus.

OBJECTIVES: The function of γ-amino butyric acid receptor (GR) was related with tinnitus. But, the effects of Spirulina platensis water extract (SP) on the mRNA expression of GRAß3 in mice with tinnitus were still unclear. METHOD: Eighteen SAMP8 mice were divided into the control group (intraperitoneal injection of saline, once per day), the tinnitus group (intraperitoneal injection of salicylate, 300 mg/kg body weight once per day), and the spirulina group [intraperitoneal injection of salicylate, 300 mg/kg body weight and oral SP supplementation (1000 mg/kg body weight) once per day]. Effects of SP on the mRNA expression of GRAß3 in the cochlea and brain of mice were studied for 4 days. RESULTS: Compared to the control group, the tinnitus group had significantly higher tinnitus scores and lower mRNA expression of GRAß3 gene in the cochlear, brainstem, hippocampus and parahippocampus, temporal lobes, and the frontal lobes. On the other hand, the spirulina group had significantly lower tinnitus scores and higher GRAß3 gene expression than the tinnitus group in all tested areas. CONCLUSION: SP could reduce salicylate-induced tinnitus possibly via increasing the salicylate-induced down-regulation of GRAß3 gene expression.

Effects of Tumor Necrosis Factor Blocker on Salicylate-Induced Tinnitus in Mice.

OBJECTIVE: Neuroinflammation is considered a novel mechanism for acute tinnitus. Here, we investigated the effects of a tumor necrosis factor (TNF) blocker on the gene expression of inflammatory-cytokine in the cochlea in a tinnitus animal model. METHODS: Enbrel® (30 mg/kg, intraperitoneally (i.p.)) were administrated to the mice with the salicylate induced tinnitus for 3 days. Tinnitus score and mRNA expression levels of TNFR1, TNFR2, and N-methyl-d-aspartate receptor subunit 2B (NR2B) and its downstream regulatory element antagonist modulator (DREAM) in the cochlea of mice were measured and compared to the control. RESULTS: The tinnitus score significantly decreased in the Enbrel® treated group. The mRNA levels of both TNFR1 and TNFR2 were significantly lower in the treatment than in the control group. The mRNA levels of NR2B and DREAM followed a similar trend. CONCLUSION: we found that treatment with 30 mg/ kg Enbrel® decreased salicylate-induced behavior associated with tinnitus and reduced the mRNA expression levels of TNFR1/R2, NR2B, and DREAM in the cochlea of mice. These findings supported the hypothesis that neuroinflammation might be a novel mechanism for salicylate-induced tinnitus.

Human Auditory Cortex Neurochemistry Reflects the Presence and Severity of Tinnitus.

It is not known why tinnitus occurs in some cases of hearing damage but not others. Abnormalities of excitation-inhibition balance could influence whether tinnitus develops and its severity if it does. Animal models of hearing damage, which also produce tinnitus based on behavioral evidence, have identified abnormalities of GABAergic inhibition, both cortically and subcortically. However, the precise relationships of GABA inhibitory changes to tinnitus itself, as opposed to other consequences of hearing damage, remain uncertain. Here, we used magnetic resonance spectroscopy to non-invasively quantify GABA in the left (LAC) and right (RAC) auditory cortices of a group of 14 patients with lateralized tinnitus (eight left ear) and 14 controls matched for age, sex, and hearing. We also explored the potential relationships with other brain metabolites (i.e., choline, N-acetylaspartate, and creatine). The presence of tinnitus was associated with a reduction in auditory cortex GABA concentration. Regardless of tinnitus laterality, post hoc testing indicated reductions that were significant in RAC and nonsignificant in LAC. Tinnitus severity and hearing loss were correlated positively with RAC choline but not GABA. We discuss the results in the context of current models of tinnitus and methodological constraints. SIGNIFICANCE STATEMENT: Permanently affecting one in seven adults, tinnitus lacks both widely effective treatments and adequate understanding of its brain mechanisms. Existing animal models represent tinnitus that may not be distinguishable from homeostatic responses to the auditory insults used to induce it. Human studies can be well controlled in this regard but are usually not (with few even matching control subjects for hearing loss) and are limited in scope as a result of relying solely on non-invasive recording techniques. Here, we exploit recent advances in non-invasive spectroscopic techniques to establish, in a human study tightly controlled for hearing loss and hyperacusis, that tinnitus is associated with a significant reduction in auditory cortex GABA concentration, which has implications for understanding and treatment of the condition.

Enhanced GABAA-Mediated Tonic Inhibition in Auditory Thalamus of Rats with Behavioral Evidence of Tinnitus.

Accumulating evidence suggests a role for inhibitory neurotransmitter dysfunction in the pathology of tinnitus. Opposing hypotheses proposed either a pathologic decrease or increase of GABAergic inhibition in medial geniculate body (MGB). In thalamus, GABA mediates fast synaptic inhibition via synaptic GABAA receptors (GABAARs) and persistent tonic inhibition via high-affinity extrasynaptic GABAARs. Given that extrasynaptic GABAARs control the firing mode of thalamocortical neurons, we examined tonic GABAAR currents in MGB neurons in vitro, using the following three groups of adult rats: unexposed control (Ctrl); sound exposed with behavioral evidence of tinnitus (Tin); and sound exposed with no behavioral evidence of tinnitus (Non-T). Tonic GABAAR currents were evoked using the selective agonist gaboxadol. Months after a tinnitus-inducing sound exposure, gaboxadol-evoked tonic GABAAR currents showed significant tinnitus-related increases contralateral to the sound exposure. In situ hybridization studies found increased mRNA levels for GABAAR δ-subunits contralateral to the sound exposure. Tin rats showed significant increases in the number of spikes per burst evoked using suprathreshold-injected current steps. In summary, we found little evidence of tinnitus-related decreases in GABAergic neurotransmission. Tinnitus and chronic pain may reflect thalamocortical dysrhythmia, which results from abnormal theta-range resonant interactions between thalamus and cortex, due to neuronal hyperpolarization and the initiation of low-threshold calcium spike bursts (Walton and Llinás, 2010). In agreement with this hypothesis, we found tinnitus-related increases in tonic extrasynaptic GABAAR currents, in action potentials/evoked bursts, and in GABAAR δ-subunit gene expression. These tinnitus-related changes in GABAergic function may be markers for tinnitus pathology in the MGB.

Expressions of ion co-transporter genes in salicylate-induced tinnitus and treatment effects of spirulina.

BACKGROUND: Although the activity of tinnitus-related ion co-transporter are known, their mRNA expressions has seldom been reported. We aimed to investigate the mRNA expressions of tinnitus-related ion co-transporter genes, and treatment effects of Spirulina. METHODS: The mRNA expressions of K(+)-Cl(-) co-transporter (KCC2) and Na-K-2Cl co-transporter 1 (NKCC1) genes in the cochlea and brain of mice were evaluated after tinnitus was induced by intraperitoneal injection of salicylate. The effects of spirulina water extract on these gene expressions were investigated. RESULTS: Compared to the control group, the tinnitus scores increased significantly, however, the salicylate-induced tinnitus could be reduced significantly by spirulina water extract. The tinnitus group had higher of borderline significance mRNA expression of KCC2 gene in the cochlear, significantly higher in the temporal lobes and in the frontal lobes. Meanwhile, compared to the tinnitus group, the spirulina group had significantly lower mRNA expression of KCC2 gene in the cochlear, temporal lobes, frontal lobes and parahippocampus/hippocampus. However, the NKCC1 mRNA expression was not significantly different between three groups in the cochlea and these brain areas. CONCLUSION: Salicylate-induced tinnitus might be associated with increased mRNA expression of KCC2 gene, but not with mRNA expressions of NKCC1 gene in the cochlear and some tinnitus-related brain areas. Spirulina reduced the expression of KCC2 genes in salicylate-induced tinnitus.

Pathogenic plasticity of Kv7.2/3 channel activity is essential for the induction of tinnitus.

Tinnitus, the perception of phantom sound, is often a debilitating condition that affects many millions of people. Little is known, however, about the molecules that participate in the induction of tinnitus. In brain slices containing the dorsal cochlear nucleus, we reveal a tinnitus-specific increase in the spontaneous firing rate of principal neurons (hyperactivity). This hyperactivity is observed only in noise-exposed mice that develop tinnitus and only in the dorsal cochlear nucleus regions that are sensitive to high frequency sounds. We show that a reduction in Kv7.2/3 channel activity is essential for tinnitus induction and for the tinnitus-specific hyperactivity. This reduction is due to a shift in the voltage dependence of Kv7 channel activation to more positive voltages. Our in vivo studies demonstrate that a pharmacological manipulation that shifts the voltage dependence of Kv7 to more negative voltages prevents the development of tinnitus. Together, our studies provide an important link between the biophysical properties of the Kv7 channel and the generation of tinnitus. Moreover, our findings point to previously unknown biological targets for designing therapeutic drugs that may prevent the development of tinnitus in humans.

Human Auditory and Adjacent Nonauditory Cerebral Cortices Are Hypermetabolic in Tinnitus as Measured by Functional Near-Infrared Spectroscopy (fNIRS).

Tinnitus is the phantom perception of sound in the absence of an acoustic stimulus. To date, the purported neural correlates of tinnitus from animal models have not been adequately characterized with translational technology in the human brain. The aim of the present study was to measure changes in oxy-hemoglobin concentration from regions of interest (ROI; auditory cortex) and non-ROI (adjacent nonauditory cortices) during auditory stimulation and silence in participants with subjective tinnitus appreciated equally in both ears and in nontinnitus controls using functional near-infrared spectroscopy (fNIRS). Control and tinnitus participants with normal/near-normal hearing were tested during a passive auditory task. Hemodynamic activity was monitored over ROI and non-ROI under episodic periods of auditory stimulation with 750 or 8000 Hz tones, broadband noise, and silence. During periods of silence, tinnitus participants maintained increased hemodynamic responses in ROI, while a significant deactivation was seen in controls. Interestingly, non-ROI activity was also increased in the tinnitus group as compared to controls during silence. The present results demonstrate that both auditory and select nonauditory cortices have elevated hemodynamic activity in participants with tinnitus in the absence of an external auditory stimulus, a finding that may reflect basic science neural correlates of tinnitus that ultimately contribute to phantom sound perception.

Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus.

Spontaneous neuronal activity in dorsal cochlear nucleus (DCN) may be involved in the physiological processes underlying salicylate-induced tinnitus. As a neuronal activity marker, immediate-early gene (IEG) expression, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) and the early growth response gene-1 (Egr-1), appears to be highly correlated with sensory-evoked neuronal activity. However, their relationships with tinnitus induced by salicylate have rarely been reported in the DCN. In this study, we assessed the effect of acute and chronic salicylate treatment on the expression of N-methyl D-aspartate receptor subunit 2B (NR2B), Arg3.1, and Egr-1. We also observed ultrastructural alterations in the DCN synapses in an animal model of tinnitus. Levels of mRNA and protein expression of NR2B and Arg3.1 were increased in rats that were chronically administered salicylate (200 mg/kg, twice daily for 3, 7, or 14 days). These levels returned to baseline 14 days after cessation of treatment. However, no significant changes were observed in Egr-1 gene expression in any groups. Furthermore, rats subjected to long-term salicylate administration showed more presynaptic vesicles, thicker and longer postsynaptic densities, and increased synaptic interface curvature. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that salicylate can cause neural plasticity changes at the DCN level.

Expression of Dopamine Receptor 1A and Cannabinoid Receptor 1 Genes in the Cochlea and Brain after Salicylate-Induced Tinnitus.

The purpose of this study was to investigate the mRNA expression of the dopamine receptor 1A (DR1A) and cannabinoid receptor 1 (CR1) genes in mice with tinnitus. Sixteen 3-month-old male SAMP8 mice were randomly and equally divided into two groups (8 mice in each group): a control (saline-treated) group and a tinnitus (salicylate-treated) group. The mRNA expression of the DR1A and CR1 genes in the cochleae and brains of the mice was evaluated after tinnitus had been induced by intraperitoneal injection of sodium salicylate (300 mg/kg body weight). The results showed that 4-day salicylate treatment (unlike 4-day saline treatment) caused a significant increase in the tinnitus score and in mRNA expression of the DR1A gene in the cochlea, the brainstem and inferior colliculus, the hippocampus and parahippocampus, and the temporal lobe, but not the frontal lobe. Conversely, 4-day salicylate treatment caused significantly lower mRNA expression of the CR1 gene in the cochlea and all the brain areas tested. In summary, salicylate-induced tinnitus may be associated with increased mRNA expression of the DR1A gene - but with decreased mRNA expression of the CR1 gene - in the cochlea and in many tinnitus-related brain areas.