Salicylate intoxication is a cause of tinnitus and comorbidly associated with anxiety in humans. In a previous work, we showed that salicylate induces anxiety-like behavior and hippocampal type 2 theta oscillations (theta2) in mice. Here we investigate if the anxiogenic effect of salicylate is dependent on age and previous tinnitus experience. We also tested whether a single dose of DMT can prevent this effect. Using microwire electrode arrays, we recorded local field potential in young (4-5-month-old) and old (11-13-month-old) mice to study the electrophysiological effect of tinnitus in the ventral hippocampus (vHipp) and medial prefrontal cortex (mPFC) in an open field arena and elevated plus maze 1 h after salicylate (300 mg/kg) injection. We found that anxietylike behavior and increase in theta2 oscillations (4-6 Hz), following salicylate pre-treatment, only occurs in young (normal hearing) mice. We also show that theta2 and slow gamma oscillations increase in the vHipp and mPFC in a complementary manner during anxiety tests in the presence of salicylate. Finally, we show that pretreating mice with a single dose of the hallucinogenic 5-MeO-DMT prevents anxiety-like behavior and the increase in theta2 and slow gamma oscillations after salicylate injection in normal hearing young mice. This work further support the hypothesis that anxiety-like behavior after salicylate injection is triggered by tinnitus and require normal hearing. Moreover, our results show that hallucinogenic compounds can be effective in treating tinnitus-related anxiety.

Salicylate intoxication is a cause of tinnitus in humans and it is often used to produce tinnitus-like perception in animal models. Here, we assess whether salicylate induces anxiety-like electrophysiological and behavioral signs. Using microwire electrode arrays, we recorded local field potential in the ventral and, in some experiments dorsal hippocampus, in an open field arena 1 hr after salicylate (300 mg/kg) injection. We found that animals treated with salicylate moved dramatically less than saline treated animals. Salicylate-treated animals showed a strong 4-6 Hz (type 2) oscillation in the ventral hippocampus (with smaller peaks in dorsal hippocampus electrodes). Coherence in the 4-6 Hz-theta band was low in the ventral and dorsal hippocampus when compared to movement-related theta coherence (7-10 Hz). Moreover, movement related theta oscillation frequency decreased and its dependency on running speed was abolished. Our results suggest that salicylate-induced theta is mostly restricted to the ventral hippocampus. Slow theta has been classically associated to anxiety-like behaviors. Here, we show that salicylate application can consistently generate low frequency theta in the ventral hippocampus. Tinnitus and anxiety show strong comorbidity and the increase in ventral hippocampus low frequency theta could be part of this association.

Background The dorsal cochlear nucleus (DCN) is a region known to integrate somatosensory and auditory inputs and is identified as a potential key structure in the generation of phantom sound perception, especially noise-induced tinnitus. Yet, how altered homeostatic plasticity of the DCN induces and maintains the sensation of tinnitus is not clear. Here, we chemogenetically decrease activity of a subgroup of DCN neurons, Ca2+/Calmodulin kinase 2 α (CaMKII α)-positive DCN neurons, using Gi-coupled human M4 Designer Receptors Exclusively Activated by Designer Drugs (hM4Di DREADDs), to investigate their role in noise-induced tinnitus. Results Mice were exposed to loud noise (9–11kHz, 90dBSPL, 1h, followed by 2h of silence), and auditory brainstem responses (ABRs) and gap prepulse inhibition of acoustic startle (GPIAS) were recorded 2 days before and 2 weeks after noise exposure to identify animals with a significantly decreased inhibition of startle, indicating tinnitus but without permanent hearing loss. Neuronal activity of CaMKII α+ neurons expressing hM4Di in the DCN was lowered by administration of clozapine-N-oxide (CNO). We found that acutely decreasing firing rate of CaMKII α+ DCN units decrease tinnitus-like responses (p = 3e −3, n = 11 mice), compared to the control group that showed no improvement in GPIAS (control virus; CaMKII α-YFP + CNO, p = 0.696, n = 7 mice). Extracellular recordings confirmed CNO to decrease unit firing frequency of CaMKII α-hM4Di+ mice and alter best frequency and tuning width of response to sound. However, these effects were not seen if CNO had been previously administered during the noise exposure (n = 6 experimental and 6 control mice). Conclusion We found that lowering DCN activity in mice displaying tinnitus-related behavior reduces tinnitus, but lowering DCN activity during noise exposure does not prevent noise-induced tinnitus. Our results suggest that CaMKII α-positive cells in the DCN are not crucial for tinnitus induction but play a significant role in maintaining tinnitus perception in mice.

The dorsal cochlear nucleus (DCN) is a region of particular interest for auditory and tinnitus research. Yet, lack of useful genetic markers for in vivo manipulations hinders elucidation of the DCN contribution to tinnitus pathophysiology. This work assesses whether adeno-associated viral vectors (AAV) containing the calcium/calmodulin-dependent protein kinase 2 alpha (CaMKIIα) promoter and a mouse line of nicotinic acetylcholine receptor alpha 2 subunit (Chrna2)-Cre can target specific DCN populations. We found that CaMKIIα cannot be used to target excitatory fusiform DCN neurons as labelled cells showed diverse morphology indicating they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 by the CaMKIIα promoter generated spikes in some units but firing rate decreased when light stimulation coincided with sound. Expression and activation of CaMKIIα-eArchaerhodopsin3.0 in the DCN produced inhibition in some units but sound-driven spikes were delayed by concomitant light stimulation. We explored the existence of Cre+ cells in the DCN of Chrna2-Cre mice by hydrogel embedding technique (CLARITY). There were almost no Cre+ cell bodies in the DCN; however, we identified profuse projections arising from the ventral cochlear nucleus (VCN). Anterograde labeling in the VCN revealed projections to the ipsilateral superior olive and contralateral medial nucleus of the trapezoid body (bushy cells); and a second bundle terminating in the DCN, suggesting the latter to be excitatory Chrna2+ T-stellate cells. Exciting Chrna2+ cells increased DCN firing. This work shows that cortical molecular tools may be useful for manipulating the DCN especially for tinnitus studies.

Aims/Hypothesis: Tinnitus is a phantom sound perception affecting both auditory and limbic structures. The mechanisms of tinnitus remain unclear and it is debatable whether tinnitus alters attention to sound and the ability to inhibit repetitive sounds, a phenomenon also known as auditory gating. Methods: 22 male C57BL/6J mice were used in this study. Anesthetized mice were exposed to a 9-11 kHz narrow band noise (90 dBSPL for 1 hr) and sham exposed mice were used as controls. Hearing thresholds were measured using auditory brainstem responses (ABRs) and tinnitus was assessed using Gap prepulse inhibition of acoustic startle (GPIAS). After the induction of tinnitus, mice were implanted multi-electrodes to assess auditory event-related potentials (aERPs) in the dorsal hippocampus in response to paired clicks. Alterations of aERPs under nicotine (1.0 mg/kg, intraperitoneal (i.p.) or cannabis extract (100 mg/Kg, i.p.) were evaluated (in isolation or in combination), the latter containing 47.25 mg/kg of tetrahydrocannabinol (THC); 0.43 mg/kg of cannabidiol (CBD) and 1.17 mg/kg of cannabinol (CBN), as analyzed by high-performance liquid chromatography (HPLC). Saline-treated animals were used as controls. Results: Our results show that mice with behavioral evidence of tinnitus display auditory gating of repetitive click, but with larger amplitudes and longer latencies of the N40 component. In contrast, no difference was observed in the P80 amplitude and latency between groups or treatments. The combination of cannabis extract and nicotine also improved auditory gating ratio in mice with noise-induced tinnitus without permanent hearing threshold shifts by strongly increasing the first N40 click amplitude but without altering the second click response amplitude. Furthermore, the increased latency of the N40 component suggests altered temporal processing of triggered attention in mice with tinnitus due to an increased sensitivity to the exposure to cannabis extract. Conclusion/Interpretation: In summary, we show that nicotine and cannabis extract alter sensory gating in mice with behavioral evidence of tinnitus and propose that the altered central plasticity in tinnitus is more sensitive to the combined actions on the cholinergic and the endocannabinoid systems. We conclude that the limbic system may play a role in the altered sensory gating responses on tinnitus since the hippocampus responses to auditory inputs are altered. These findings could enable a new understanding of which neuronal pathways could be involved in sensory gating in tinnitus.