Unraveling the role of Slc10a4 in auditory processing and sensory motor gating: Implications for neuropsychiatric disorders?

BACKGROUND: Psychiatric disorders, such as schizophrenia, are complex and challenging to study, partly due to the lack of suitable animal models. However, the absence of the Slc10a4 gene, which codes for a monoaminergic and cholinergic associated vesicular transporter protein, in knockout mice (Slc10a4-/-), leads to the accumulation of extracellular dopamine. A major challenge for studying schizophrenia is the lack of suitable animal models that accurately represent the disorder. We sought to overcome this challenge by using Slc10a4-/- mice as a potential model, considering their altered dopamine levels. This makes them a potential animal model for schizophrenia, a disorder known to be associated with altered dopamine signaling in the brain. METHODS: The locomotion, auditory sensory filtering and prepulse inhibition (PPI) of Slc10a4-/- mice were quantified and compared to wildtype (WT) littermates. Intrahippocampal electrodes were used to record auditory event-related potentials (aERPs) for quantifying sensory filtering in response to paired-clicks. The channel above aERPs phase reversal was chosen for reliably comparing results between animals, and aERPs amplitude and latency of click responses were quantified. WT and Slc10a4-/- mice were also administered subanesthetic doses of ketamine to provoke psychomimetic behavior. RESULTS: Baseline locomotion during auditory stimulation was similar between Slc10a4-/- mice and WT littermates. In WT animals, normal auditory processing was observed after i.p saline injections, and it was maintained under the influence of 5 mg/kg ketamine, but disrupted by 20 mg/kg ketamine. On the other hand, Slc10a4-/- mice did not show significant differences between N40 S1 and S2 amplitude responses in saline or low dose ketamine treatment. Auditory gating was considered preserved since the second N40 peak was consistently suppressed, but with increased latency. The P80 component showed higher amplitude, with shorter S2 latency under saline and 5 mg/kg ketamine treatment in Slc10a4-/- mice, which was not observed in WT littermates. Prepulse inhibition was also decreased in Slc10a4-/- mice when the longer interstimulus interval of 100 ms was applied, compared to WT littermates. CONCLUSION: The Slc10a4-/- mice responses indicate that cholinergic and monoaminergic systems participate in the PPI magnitude, in the temporal coding (response latency) of the auditory sensory gating component N40, and in the amplitude of aERPs P80 component. These results suggest that Slc10a4-/- mice can be considered as potential models for neuropsychiatric conditions.

Alterations of auditory sensory gating in mice with noise-induced tinnitus treated with nicotine and cannabis extract.

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. Here we investigate if noise exposure interferes with auditory gating and whether natural extracts of cannabis or nicotine could improve auditory pre-attentional processing in noise-exposed mice. We used 22 male C57BL/6J mice divided into noise-exposed (exposed to a 9-11 kHz narrow band noise for 1 h) and sham (no sound during noise exposure) groups. Hearing thresholds were measured using auditory brainstem responses, and tinnitus-like behavior was assessed using Gap prepulse inhibition of acoustic startle. After noise exposure, mice were implanted with multi-electrodes in the dorsal hippocampus to assess auditory event-related potentials in response to paired clicks. The results showed that mice with tinnitus-like behavior displayed auditory gating of repetitive clicks, but with larger amplitudes and longer latencies of the N40 component of the aERP waveform. The combination of cannabis extract and nicotine improved the auditory gating ratio in noise-exposed mice without permanent hearing threshold shifts. Lastly, the longer latency of the N40 component appears due to an increased sensitivity to cannabis extract in noise-exposed mice compared to sham mice. The study suggests that the altered central plasticity in tinnitus is more sensitive to the combined actions on the cholinergic and the endocannabinoid systems. Overall, the findings contribute to a better understanding of pharmacological modulation of auditory sensory gating.

Loud noise-exposure changes the firing frequency of subtypes of layer 5 pyramidal neurons and Martinotti cells in the mouse auditory cortex.

Introduction: Loud noise-exposure can generate noise-induced tinnitus in both humans and animals. Imaging and in vivo studies show that noise exposure affects the auditory cortex; however, cellular mechanisms of tinnitus generation are unclear. Methods: Here we compare membrane properties of layer 5 (L5) pyramidal cells (PCs) and Martinotti cells expressing the cholinergic receptor nicotinic alpha 2 subunit gene (Chrna2) of the primary auditory cortex (A1) from control and noise-exposed (4-18 kHz, 90 dB, 1.5 h, followed by 1.5 h silence) 5-8 week old mice. PCs were furthermore classified in type A or type B based on electrophysiological membrane properties, and a logistic regression model predicting that afterhyperpolarization (AHP) and afterdepolarization (ADP) are sufficient to predict cell type, and these features are preserved after noise trauma. Results: One week after a loud noise-exposure no passive membrane properties of type A or B PCs were altered but principal component analysis showed greater separation between type A PCs from control and noise-exposed mice. When comparing individual firing properties, noise exposure differentially affected type A and B PC firing frequency in response to depolarizing current steps. Specifically, type A PCs decreased initial firing frequency in response to +200 pA steps (p = 0.020) as well as decreased steady state firing frequency (p = 0.050) while type B PCs, on the contrary, significantly increased steady state firing frequency (p = 0.048) in response to a + 150 pA step 1 week after noise exposure. In addition, L5 Martinotti cells showed a more hyperpolarized resting membrane potential (p = 0.04), higher rheobase (p = 0.008) and an increased initial (p = 8.5 × 10-5) and steady state firing frequency (p = 6.3 × 10-5) in slices from noise-exposed mice compared to control. Discussion: These results show that loud noise can cause distinct effects on type A and B L5 PCs and inhibitory Martinotti cells of the primary auditory cortex 1 week following noise exposure. As the L5 comprises PCs that send feedback to other areas, loud noise exposure appears to alter levels of activity of the descending and contralateral auditory system.

Decreasing dorsal cochlear nucleus activity ameliorates noise-induced tinnitus perception in mice.

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.

Using Cortical Neuron Markers to Target Cells in the Dorsal Cochlear Nucleus.

The dorsal cochlear nucleus (DCN) is a region of particular interest for auditory and tinnitus research. However, 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 2α (CaMKIIα) promoter and a mouse line of nicotinic acetylcholine receptor α2 subunit (Chrna2)-Cre can target specific DCN populations. We found that CaMKIIα cannot be used to target excitatory fusiform DCN neurons as labeled cells showed diverse morphology indicating they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 (ChR2) 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 (MNTB; 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.

Anxiety-like behavior induced by salicylate depends on age and can be prevented by a single dose of 5-MeO-DMT.

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 1h after salicylate (300mg/kg) injection. We found that anxiety-like 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 pre-treating 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 induces anxiety-like behavior and slow theta oscillation and abolishes the relationship between running speed and fast theta oscillation frequency.

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.