Thalamo-cortical neural mechanism of sodium salicylate-induced hyperacusis and anxiety-like behaviors
https://ror.org/00v408z34grid.254145.30000 0001 0083 6092Department of Physiology, School of Life Sciences, China Medical University, Shenyang, China
https://ror.org/00v408z34grid.254145.30000 0001 0083 6092Laboratory of Hearing Research, School of Life Sciences, China Medical University, Shenyang, China
grid.412467.20000 0004 1806 3501Department of Nephrology, Shengjing Hospital of China Medical University, Shenyang, China
grid.412449.e0000 0000 9678 1884Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China
https://ror.org/04wjghj95grid.412636.4Department of Rheumatology and Immunology, The First Hospital of China Medical University, Shenyang, China
Abstract
Tinnitus has been identified as a potential contributor to anxiety. Thalamo-cortical pathway plays a crucial role in the transmission of auditory and emotional information, but its casual link to tinnitus-associated anxiety remains unclear. In this study, we explore the neural activities in the thalamus and cortex of the sodium salicylate (NaSal)-treated mice, which exhibit both hyperacusis and anxiety-like behaviors. We find an increase in gamma band oscillations (GBO) in both auditory cortex (AC) and prefrontal cortex (PFC), as well as phase-locking between cortical GBO and thalamic neural activity. These changes are attributable to a suppression of GABAergic neuron activity in thalamic reticular nucleus (TRN), and optogenetic activation of TRN reduces NaSal-induced hyperacusis and anxiety-like behaviors. The elevation of endocannabinoid (eCB)/ cannabinoid receptor 1 (CB1R) transmission in TRN contributes to the NaSal-induced abnormalities. Our results highlight the regulative role of TRN in the auditory and limbic thalamic-cortical pathways.
Web Summary
Sodium salicylate enhances endocannabinoid signaling, reducing GABAergic activity in the thalamic reticular nucleus. This increases gamma oscillations in the auditory cortex and prefrontal cortex, causing hyperacusis and anxiety-like behaviors.
Introduction
Anxiety disorders rank among the most prevalent and debilitating mental health conditions worldwide1,2, with a global prevalence between 3.8% and 25%3,4. Stressful life events are a major contributor to anxiety disorders5, and tinnitus, the phantom perception of sound without a corresponding external acoustic stimulus, is a common underlying factor for stress. Persistent tinnitus can cause a state of chronic stress and the stress can further aggravate tinnitus in turn6,7. Several clinical studies have indicated that individuals with tinnitus exhibit higher rates of anxiety disorders compared to the general population8–11. Additionally, the lifetime incidence of tinnitus significantly increases in individuals with anxiety disorders12. Although the link between tinnitus as a stressful factor and anxiety disorders was clear, the mechanism underlying this association has remained elusive.
Tinnitus is often triggered by hearing damage, but cochlear ablation or auditory nerve section never eradicates the perception of tinnitus13. This suggests that the generation of tinnitus involves a central mechanism. Previous studies have shown that tinnitus patients have stronger gamma band oscillation (GBO) in the auditory cortex (AC)14, which were associated with a reduction in the transmission of the major inhibitory neurotransmitter gamma-aminobutyric acid (GABA)15. In anxiety disorder patients, neural activity in the prefrontal cortex (PFC) was found to be higher during states of mental tension16,17. Although current data indicate the involvement of AC in tinnitus and the importance of PFC in anxiety, intensive research is still needed to address the mechanism behind the comorbidity of tinnitus and anxiety.
The thalamus traditionally functions as a ‘relay station’ for most sensory inputs on their way to cerebral cortex18,19, but recent studies suggest it also plays an active role in processing and integrating information20. Therefore, the auditory signals associated with tinnitus and the emotional signals associated with anxiety have to be processed by the thalamus before being transmitted to the cerebral cortex. The thalamus comprises two types of nuclei: first-order and higher-order thalamic nuclei21,22. First-order thalamic nuclei relay bottom-up sensory information to primary sensory, visual and auditory cortices23,24, of which the medial geniculate body (MGB) relays neural information about sound to the AC25. Higher-order nuclei receive their principal driver inputs from cortical layer V and transfer information among functionally related cortical regions21,22,26, of which the mediodorsal thalamus (MD), the largest thalamic input to the prefrontal cortex (PFC), is critical for maintaining cognitive control signals underlying emotion27, attention28, cognitive29, adaptive decision making30 and working memory31. The thalamic reticular nucleus (TRN), a sheet of GABAergic neurons surrounding the dorsal and lateral portions of the thalamus, provides the major inhibitory input to thalamocortical neurons (including the MGB and MD)32–37. As the gatekeeper of information transfer from the thalamus to the cortex, the TRN regulates information flow to maintain neural network balance and is implicated in sensory detection38,39, attention40–42, and arousal43–45. Nonetheless, due to the limited spatial resolution of non-invasive electroencephalogram (EEG) and functional magnetic resonance imaging (fMRI), there is insufficient evidence concerning any abnormal activity in the MGB, MD, and TRN in patients with tinnitus or anxiety disorders. Thus, despite the strong connection between the cortex and thalamus, it remains unclear whether the abnormal cortical neural activity related to tinnitus and anxiety originates from the thalamus.
To address this issue, it is necessary to conduct neurophysiological experiments on animal models of tinnitus. Sodium salicylate (NaSal)-induced tinnitus animal models have become a popular and valuable tool for pharmacological and pathological studies of the auditory system46–48, due to the potential of NaSal to cause adverse reactions like tinnitus and hyperacusis in humans47,49–51. These studies have revealed an increase in neuronal activity and GBO in both the MGB and AC, along with enhanced functional connectivity between them during NaSal-induced tinnitus52–56. Additionally, a reduction in presynaptic releases of GABA was reported in the AC of NaSal-treated rats57. Moreover, NaSal induces anxiety-like behaviors in mice, which may be associated with profound electrophysiological changes in the limbic system, such as PFC, lateral amygdala, and ventral hippocampus58–60. However, the specific neural mechanisms underlying the tinnitus associated anxiety in NaSal model mice remain unclear.
The primary objective of our study was to investigate the neural and molecular mechanisms underlying hyperacusis and anxiety-like behaviors of NaSal-treated mice using in vivo electrophysiological recording, fiber-optic recording, targeted recombination in active populations (TRAP), immunofluorescence, optogenetics, conditional gene knockout, and behavior tests. We found evidence supporting the idea that NaSal-induced abnormalities of auditory function and emotional state are due to the reduction in GABAergic neuronal activity in TRN, which leads to the hyperactivity of MGB-AC and MD-PFC. Moreover, an abnormal level of endocannabinoids (eCBs) acting on cannabinoid receptor 1 (CB1R) of GABAergic neurons in TRN may underly the NaSal-induced presynaptic dysfunction of TRN neurons.
Results
NaSal treatment induces hyperacusis and anxiety-like behaviors
Firstly, we performed the prepulse inhibition (PPI) test to investigate whether NaSal-treated mice exhibited hyperacusis (Fig. 1A). NaSal-treated mice significantly exhibited an increased acoustic startle response (ASR, i.e., startle amplitude) when tested with the acoustic stimuli at the sound pressure level of 120 dB (Fig. 1B, t = 19.49, df = 28, p < 0.0001). Analysis of %PPI of the ASR after the presentation of 80, 85, 90, 95 and 100 dB prepulse acoustic stimuli showed a main treatment effect (Fig. 1C, F (1, 140) = 648.90, p < 0.0001), with NaSal-treated mice displaying more PPI than vehicle-treated mice, and a main effect of prepulse intensity (F (4, 140) = 37.95, p < 0.0001). No significant treatment × prepulse intensity interaction was found (F (4, 140) = 0.20, p = 0.9393).
Anxiety-like behavior in mice of different treatments was assessed using the open field test (OFT, Fig. 1D–G) and elevated O-maze test (O-maze, Fig. 1H–L). NaSal-treated mice spent significantly less time in the center of the arena compared to the vehicle-treated mice (Fig. 1F, t = 5.17, df = 28, p < 0.0001). Additionally, these mice exhibited a reduced traveled distance. (Fig. 1G, t = 5.32, df = 28, p < 0.0001). In the O-maze, NaSal-treated mice displayed decreased time spent in open arms (Fig. 1J, t = 4.04, df = 28, p = 0.0004), and fewer entries into open arms (Fig. 1K, t = 4.71, df = 28, p < 0.0001).
It’s worthy to note that the reduction of exploration may be due to hypo-locomotion, rather than anxious state. To validate it, we measured traveled distance of spontaneous activity in the home cage of mouse. No significant difference was found between the vehicle- and NaSal-treated groups. Because the OFT and O-maze test represented a novel environment, the reduction of traveled distance measured here reflects the reduction of novelty-induced locomotion rather than spontaneous locomotion. Therefore, our results of OFT and O-maze may reflect an anxiety-like behavior induced by NaSal treatment.
NaSal treatment enhances Fos expression and GBO of AC and PFC
Next, we examined the effect of NaSal treatment on AC and PFC neurons using TRAP technology for the immediate early gene product Fos (Fig. 2A). NaSal treatment significantly increased the percentage of FosTRAPed neurons (i.e., NeuN+ FosTRAPed cells/ total mature NeuN+ cells) in the AC (Fig. 2B–D, t = 8.04, df = 18, p < 0.0001) and PFC (Fig. 2E–G, t = 16.15, df = 18, p < 0.0001). We selected these Fos-positive regions as targets for subsequent experiments investigating the AC and PFC.
We further recorded the local field potentials (LFPs) in the AC and PFC from 0.5 h pre- to 3 h post-vehicle/NaSal injection (Fig. 2H, I). The temporal-spectrograms of LFP exhibit an increase of GBO power after NaSal injection (Fig. 2J, L). The mean GBO power was significantly increased in the AC from 0 to 1.5 h after NaSal injection (Fig. 2K, F treatment (1, 28) = 21.10, p < 0.0001, F time (6, 168) = 38.44, p < 0.0001, F treatment × time interaction (6, 168) = 30.34, p < 0.0001), and in the PFC from 0.5 to 2 h (Fig. 2M, F treatment (1, 28) = 36.53, p < 0.0001, F time (6, 168) = 34.45, p < 0.0001, F treatment × time interaction (6, 168) = 27.71, p < 0.0001). Thus, NaSal can induce neural activation and GBO increase in the AC and PFC.
Effects of NaSal treatment on the activities of thalamus and their phase-locking with cortical GBO bursts
To further investigate the effect of NaSal on neural activities, we used fiber photometry to record the immediate calcium signals from MGB, MD, and TRN neurons (Fig. 3A). We injected rAAV-CaMKIIa-GCaMP6s into either the unilateral MGB or MD of WT mice. While CaMKIIa may also be expressed in inhibitory neurons, previous histological studies have demonstrated that the sensory thalamus of rodents is primarily composed of glutamatergic neurons, with a very small proportion of inhibitory neurons32,61,62. Therefore, the use of the CaMKIIa promoter to transfect the glutamatergic neurons of MGB and MD is appropriate. On the other hand, the TRN area is the sole region in the rodent thalamus where GABAergic inhibitory neurons aggregate63. We used rAAV-CAG-DIO-GCaMP6s injected into TRN of GAD2-cre mice to selectively transfect GABAergic neurons with GCaMP6s. After 21 days of recovery, the fiber-optic recordings were conducted at pre- and 1 h post-vehicle/NaSal injection (when NaSal-induced GBO was the most obvious). In the glutamatergic neurons of MGB, we found a significant increase in the amplitude and frequency of calcium transients between pre- and post-NaSal injection groups (Fig. 3C, t = 4.10, df = 18, p = 0.0007; Fig. 3D, t = 2.83, df = 18, p = 0.0112), but not between pre- and post-vehicle injection groups (p = 0.7853 and p = 0.4845). Similarly, the calcium transient amplitude and frequency of glutamatergic neurons in the MD was also increased after the NaSal injection (Fig. 3F, t = 2.98, df = 18, p = 0.0080; Fig. 3G, t = 2.38, df = 18, p = 0.0289). However, NaSal injection decreased the calcium transient amplitude and frequency of the TRN GABAergic neurons significantly (Fig. 3I, t = 5.10, df = 18, p < 0.0001; Fig. 3J, t = 3.74, df = 18, p = 0.0015).
Comparing with GBO bursts in the simultaneously recorded LFPs, we observed that NaSal injection enhanced the phase-locking between the GBO bursts of AC and the calcium transients of MGB neurons (Fig. 3K and L, t = 2.90, df = 18, p = 0.0096). The phase-locking between the GBO bursts of PFC and the calcium transients of MD neurons also increased significantly after NaSal injection (Fig. 3M and N, t = 4.53, df = 18, p = 0.0003). Therefore, the neural activities were decreased in the TRN by NaSal, but increased in the MGB and MD, contributing to the enhancement of GBO in the AC and PFC.
Calcium activities of MD neurons during O-maze test
Next, to examine the association between MD neuron activity and anxiety, we conducted fiber photometry in the O-maze apparatus. We analyzed calcium signals from MD glutamatergic neurons as the mice ran across different arm compartments of the O-maze. Calcium signals of vehicle-treated mice increased when transitioning from open arms to closed arms (OA → CA), while decreased, from closed arms to open arms (CA → OA, Fig. 4A, B). Compared to vehicle-treated mice, NaSal-treated mice exhibited a more pronounced increase in calcium signals at OA → CA, but a less pronounced decrease at CA → OA. Quantitative analysis showed that the area under the curve (AUC) of calcium signals at OA → CA was significantly higher in NaSal-treated mice compared to vehicle-treated mice (Fig. 4C, t = 3.05, df = 28, p = 0.0050). Conversely, at CA → OA, NaSal-treated mice showed a significantly smaller reduction in AUC compared to vehicle-treated mice (t = 2.52, df = 28, p = 0.0176).
Optogenetic activation of MGB-AC or MD-PFC induces changes in cortical GBO and alterations in auditory function or emotional state
To further investigate the association between thalamic neural activity and cortical GBO, we recorded the LFPs of the AC or PFC in response to the optogenetic activation of MGB-AC (Fig. 5A-D) or MD-PFC (Fig. 5E–H). As illustrated by Fig. 5B, F, the rhythmic activation (470 nm, 32 Hz, 5 ms pulse width) of MGB-AC and MD-PFC effectively induced resonant LFP activity in the AC and PFC, respectively. The power spectrum of the LFP exhibited a peak around 32 Hz (Fig. 5C, G), and the GBO power increased with stimulation intensity (Fig. 5D, H).
We further examined whether optogenetic activation of the MGB-AC and MD-PFC pathways could acutely induce abnormalities in auditory and emotional function using the experimental procedures outlined in Fig. 5I, N. When the MGB-AC was optogenetically activated, mice exhibited an increase in ASR intensity to acoustic stimuli at 120 dB (Fig. 5J, t = 10.75, df = 18, p < 0.0001). %PPI showed a main treatment effect (Fig. 5C, F (1, 90) = 416.30, p < 0.0001), as well as a main effect of prepulse intensity (F (4, 90) = 28.21, p < 0.0001), but no significant treatment × prepulse intensity interaction (F (4, 90) = 1.20, p = 0.3169). Conversely, activation of the MD-PFC did not induce any changes in ASR intensity or %PPI (Fig. 5L, t = 0.24, df = 18, p = 0.8098; Fig. 5M, F treatment (1, 90) = 0.50, p = 0.4819, F prepulse intensity (4, 90) = 41.82, p < 0.0001, F treatment × prepulse intensity interaction (4, 90) = 0.81, p = 0.5249). MGB-AC activation did not significantly affect time spent in open arms, open arm entry times, or self-grooming time in the O-maze (Fig. 5O, P, F (1.76, 15.84) = 0.15, p = 0.8400; Fig. 5Q, F (1.97, 17.76) = 0.89, p = 0.4286; Fig. 5R, F (2.00, 17.97) = 0.43, p = 0.6561). However, statistical analysis revealed a significant decrease in time spent in open arms and open arm entry times, and an increase in self-grooming time, a parameter relevant for high level of anxiety64,65, upon activation of the MD-PFC pathway (Fig. 5S and T, F (1.85, 16.67) = 20.41, p < 0.0001; Fig. 5U, F (1.97, 17.72) = 10.55, p = 0.0010; Fig. 5V, F (1.78, 16.03) = 25.91, p < 0.0001). These anxiety-like behaviors persisted during the Light OFF period. Thus, MGB-AC and MD-PFC activation were closely related to hyperacusis and anxiety-like behaviors, respectively.
Optogenetic activation of TRN neurons ameliorated NaSal-induced cortical GBO, hyperacusis, and anxiety-like behaviors
Next, we conducted LFP recordings on the AC and PFC of NaSal-treated mice expressing ChR2 or EYFP in the TRN GABAergic neurons to investigate the effect of optogenetic activation of TRN neurons (Fig. 6A). The results confirmed that TRN neuron activation significantly suppressed GBO in both the AC and PFC at 1-1.5 h post NaSal injection compared to the EYFP group (Fig. 6B, C, F treatment (1, 18) = 7.00, p = 0.0164, F time (4, 72) = 83.79, p < 0.0001, F treatment × time interaction (4, 72) = 54.19, p < 0.0001; Fig. 6D, E, F treatment (1, 18) = 7.12, p = 0.0156, F time (4, 72) = 102.90, p < 0.0001, F treatment × time interaction (4, 72) = 31.16, p < 0.0001).
To explore the effect of TRN neural activity on auditory function, we injected mice with NaSal and subjected them to optogenetic activation of the TRN neurons and ASR/PPI tests at 1 h after the injection (Fig. 6F). Compared to the Light OFF group, NaSal-treated mice in the Light ON group exhibited significant decreases in ASR intensity and %PPI (Fig. 6G, t = 10.56, df = 18, p < 0.0001; Fig. 6H, F treatment (1, 90) = 184.10, p < 0.0001; F prepulse intensity (4, 90) = 22.42, p < 0.0001; F treatment × prepulse intensity interaction (4, 90) = 1.41, p = 0.2383). O-maze was also performed on the NaSal-treated mice with or without activation of the TRN neurons (Fig. 6I). We found that the mice spent more time in the open arms and entered the open arms more frequently when their TRN neurons were activated (Fig. 6J and K, t = 4.23, df = 18, p = 0.0005; Fig. 6L, t = 2.37, df = 18, p = 0.0291).
NaSal treatment increases the level of eCBs in MGB and MD
Previous studies have shown that NaSal inhibits the activity of cyclooxygenase-2 (COX-2)66, which has the ability to metabolize eCBs67 (Fig. 7A). Therefore, NaSal treatment may elevate eCB level. We examined eCB dynamics in the MGB and MD using fiber-optic recording of eCB2.068 to test this hypothesis (Fig. 7B-G). Figure 7B illustrates the representative eCB2.0 fluorescence dynamics in the MGB before and after NaSal injection. No significant change was found in the amplitude and frequency of eCB2.0 transients between pre- and 1 h post-vehicle injection (Fig. 7C, t = 0.93, df = 8, p = 0.3774; Fig. 7D, t = 0.06, df = 8, p = 0.9574). However, we observed a significant increase in the amplitude and frequency of the eCB2.0 transients after NaSal injection (Fig. 7C, t = 5.36, df = 8, p = 0.0007; Fig. 7D, t = 3.32, df = 8, p = 0.0105). Similarly, the amplitude and frequency of the eCB2.0 transients in the MD were not changed by vehicle injection (Fig. 7E and F, t = 0.57, df = 8, p = 0.5857; Fig. 7G, t = 0.09, df = 8, p = 0.9338), but showed a significant increase after NaSal injection (Fig. 7F, t = 2.72, df = 8, p = 0.0263; Fig. 7G, t = 3.02, df = 8, p = 0.0165).
In the central nervous system (CNS), eCBs generally bind to presynaptic CB1Rs to suppress neurotransmitter release69. We examined the mouse database in the Allen Brain Atlas and confirmed high mRNA expression of Cnr1 (the gene encoding CB1R) in the TRN (http://mouse.brain-map.org). By using fluorescence in situ hybridization (FISH) combined with immunofluorescence in the TRN section, we found that Cnr1 mRNA was localized in cells expressing GAD67 protein (coded by Gad1 gene, Fig. 7H). Therefore, NaSal treatment may increase the level of eCBs in the MGB and MD, which can act on CB1R on TRN GABAergic neurons, mediating the presynaptic inhibition of GABA.
CB1R antagonist AM251 reduced GBO and anxiety-like behaviors induced by NaSal
To investigate whether NaSal induced GBO increase and anxiety-like behaviors through a CB1R-dependent mechanism, we repeated the LFP recordings and behavioral tests combining AM251 (CB1R antagonist) or vehicle (saline) with NaSal injection. In the LFP recording experiments (Fig. 8A–E), the mean GBO power in the AC decreased significantly from 0.5 to 1.5 h after NaSal injection in the mice with AM251 treatment (Fig. 8C, F treatment (1, 18) = 2.44, p = 0.1356, F time (4, 72) = 26.28, p < 0.0001, F treatment × time interaction (4, 72) = 9.05, p < 0.0001). Similar results were also observed in the PFC (Fig. 8E, F treatment (1, 18) = 2.80, p = 0.1118, F time (4, 72) = 12.49, p < 0.0001, F treatment × time interaction (4, 72) = 6.18, p = 0.0002).
In the ASR/PPI tests (Fig. 8F-H), AM251 treatment significantly decreased the ASR intensity in the NaSal mice, compared with vehicle treatment (Fig. 8G, t = 5.87, df = 18, p < 0.0001). %PPI was also decreased by the AM251 treatment (Fig. 8H, F treatment (1, 90) = 91.28, p < 0.0001, F prepulse intensity (4, 90) = 29.86, p < 0.0001, F treatment × prepulse interaction (4, 90) = 0.35, p = 0.8404). In the OFT (Fig. 8I-L), the mice received AM251 treatment spent significantly more time in the center of the arena (Fig. 8J and K, t = 3.49, df = 18, p = 0.0026) and traveled a longer distance compared to the vehicle-treated mice (Fig. 8L, t = 3.13, df = 18, p = 0.0058). One point should be clarified that mice in the vehicle group, which received both NaSal and vehicle treatments, exhibited more pronounced anxiety-like behaviors (reduced center time and total distance traveled) compared to those receiving a single NaSal treatment as shown in Fig. 1. This was likely due to being handled and injected twice within a short period. Both the AM251 and vehicle groups underwent similar handling and injection procedures twice during this experiment, thus this procedure would not produce any systemic interference on our observations of the effects of AM251. In the O-maze (Fig. 8M-P), the AM251-treated mice spent more time in the open arms (Fig. 8N and O, t = 5.10, df = 18, p < 0.0001) and entered more into the open arms (Fig. 8P, t = 6.52, df = 18, p < 0.0001). Therefore, CB1R has mediated the GBO increase of the AC and PFC and the anxiety-like behaviors induced by NaSal.
Selective knockout of CB1R in TRN neurons alleviated NaSal-induced enhancement of GBOs and anxiety-like behaviors
Considering that NaSal or AM251 may also impact local inhibitory circuits in brain regions such as the AC, PFC, or amygdala, it is necessary to further investigate the extent to which TRN CB1R plays a role in NaSal-induced GBO enhancement and behavioral abnormalities. For this, we injected rAAV-mDLx-CRE-EGFP into the TRN of CB1f mice to selectively knock out CB1Rs (TRN-CB1RKO), and rAAV-mDLx-EGFP was used as a control (TRN-EGFP, Fig. 9A). NaSal-treated TRN-EGFP mice showed significantly prolonged GBO enhancement in the AC and PFC compared to vehicle-treated TRN-EGFP mice. In contrast, NaSal-treated TRN-CB1RKO mice did not exhibit a pronounced enhancement in GBO (Fig. 9B-E, AC: F treatment (2, 27) = 11.26, p = 0.0003, F time (4, 108) = 14.60, p < 0.0001, F treatment × time interaction (8, 108) = 7.11, p < 0.0001; PFC: F treatment (2, 27) = 11.66, p = 0.0002, F time (4, 108) = 51.50, p < 0.0001, F treatment × time interaction (8, 108) = 43.49, p < 0.0001). Compared to vehicle-treated TRN-EGFP mice, NaSal-treated TRN-EGFP mice exhibited obvious increases in startle amplitude and %PPI in the ASR/PPI test (Fig. 9F and G), time spent in the center and total distance traveled in the OFT (Fig. 9H-J), time spent in the open arms and number of entries into the open arms in the O-maze test (Fig. 9K-M). On the contrary, there was no significant difference between NaSal-treated TRN-CB1RKO mice and vehicle-treated TRN-EGFP mice (Fig. 9F, F (2, 27) = 251.50, p < 0.0001; Fig. 9G, F treatment (2, 135) = 293.40, p < 0.0001, F prepulse intensity (4, 135) = 48.09, p < 0.0001, F treatment × prepulse intensity interaction (8, 135) = 0.64, p = 0.7409; Fig. 9I, F (2, 27) = 8.19, p = 0.0017; Fig. 9J, F (2, 27) = 4.74, p = 0.0172; Fig. 9L, F (2, 27) = 16.85, p < 0.0001; Fig. 9M, F (2, 27) = 22.29, p < 0.0001). These results suggest that CB1R within the TRN plays a critical role in the cortical GBO enhancement and behavioral alterations induced by NaSal.
Discussion
In this study, we investigated the neural and molecular mechanisms underlying hyperacusis and anxiety-like behaviors in NaSal-treated mice using behavioral tests, electrophysiological recording, fiber-optic recording, optogenetics, conditional gene knockout, and histological methods. We found the mice who underwent NaSal treatment exhibited hyperacusis and anxiety-like behaviors. This was accompanied by an increase in Fos expression and enhancement of GBO in both the AC and PFC. Additionally, NaSal increased the activity of glutamatergic neurons in the MGB and MD, as well as their phase-locking with the gamma bursts in the AC and PFC. By manipulating the MGB-AC and MD-PFC circuits, as well as TRN neurons, we could regulate the hyperacusis and anxiety-like behaviors. Further analysis revealed an elevation in the levels of eCBs in the MGB and MD of NaSal-treated mice, which acted on CB1Rs of TRN GABAergic neurons. The administration of AM251 reduced NaSal-induced GBO and behavioral abnormalities. The selective knockout of CB1Rs of TRN neurons also prevented NaSal-induced enhanced GBO and anxiety-like behaviors. These results emphasize the significance of eCBs/CB1Rs in GABAergic presynaptic inhibition and highlight the involvement of thalamic-cortical neural hyperactivation in hyperacusis and anxiety-like behaviors.
Previous studies have shown that NaSal has the ability to induce auditory perceptual disturbances (hyperacusis, tinnitus) in animals70,71. We validated the presence of these auditory abnormalities in mice by the ASR/PPI test. The NaSal-induced auditory abnormalities have been attributed to an increase in sound-evoked neural activity in the auditory processing centers, especially in the MGB and AC70,72. In addition, there is a suggested correlation between GBO in the auditory cortex and the presence of tinnitus73. GBO is an oscillatory pattern of neural activity commonly found in several brain regions74–79, and is thought to play an important role in sensory processing, attention, and memory80–82. In this study, we found that NaSal treatment enhances GBO in the AC. By the simultaneous electrophysiological recording of LFP and fiber-optic recording of neuronal calcium activity, we further found that NaSal injection enhanced the phase-locking between GBO bursts in the AC and calcium transients in the MGB neurons. Besides that, our optogenetic experiments confirmed that the activation of MGB-AC can induce hyperacusis in the ASR/PPI test, with no significant changes found in the O-maze test. In summary, NaSal-induced GBO increase in the AC is likely associated with the hyperactivation of MGB neurons, and the hyperexcitation of MGB-AC may further trigger hyperacusis in the mice.
Apart from causing transient hyperacusis and tinnitus, previous research has also documented that NaSal induces anxiety-like behaviors in animals by OFT and elevated plus maze tests59,60. Our current study also confirmed this with the usage of OFT and O-maze tests. The reason for NaSal-induced anxiety-like behaviors is thought to be associated with the increase of GBO in the limbic system, such as the PFC60. We verified that NaSal treatment amplified GBO in the PFC and enhanced phase-locking between GBO bursts in the PFC and calcium transients in the MD neurons. Fiber photometry in the O-maze apparatus further revealed an association between MD neuron activity and anxiety. In contrast to MGB-AC activation, activating MD-PFC can induce anxiety-like behaviors in the O-maze test, but no significant changes observed in the ASR/PPI test. Thus, NaSal-induced GBO increase in the PFC may be caused by the hyperactivity of MD neurons, and this could ultimately induce anxiety-like behaviors in the mice.
The prevailing view suggests that NaSal causes tinnitus or anxiety by disrupting the balance between excitatory and inhibitory neuronal activity in the brain. The inhibitory effect mediated by GABA is crucial for maintaining a balance of excitation and inhibition in the CNS83,84. Clinical experiments have indicated that the level of GABA in the AC is significantly reduced in patients with tinnitus15. In patients with anxiety disorders, GABA concentration is also reduced in the limbic system, including the PFC and anterior cingulate cortex85. Patch clamp experiments conducted on NaSal mice found decreased inhibitory currents in AC neurons57, indicating that the hyperexcitation may be caused by a reduction in GABAergic inhibitory function. Since the TRN is entirely composed of GABAergic neurons which provide inhibitory projections to several thalamic nuclei, including MGB and MD, alterations in neural activity within TRN may be a significant contributing factor to NaSal-induced inhibition of the GABA system. Our fiber photometry experiments revealed that NaSal injection led to an inhibition of GABAergic neuronal activity in the TRN, which may contribute to the hyperexcitation of MGB and MD. Furthermore, optogenetic activation of TRN GABAergic neurons suggests that increased inhibitory input from the TRN can result in the downregulation of cortical GBO and amelioration of the associated auditory and emotional abnormalities induced by NaSal. Therefore, the TRN may play a key role in modulating the functions of auditory and emotional neural circuits.
Because NaSal is an inhibitor of COX-2, which can degrade eCBs66, NaSal treatment might cause an increase in eCB levels. Our results from fiber-optic recordings of eCB2.0 fluorescence dynamics confirmed this in the MGB and MD. eCBs are signaling lipids that activate cannabinoid receptors86–89. Cannabinoid receptors are divided into two classes: CB1R, which is located primarily in the brain90,91, and cannabinoid 2 receptors (CB2R), which is located mainly in immune cells in the periphery92. In the CNS, CB1R is most abundant on certain GABAergic interneurons in regions such as the cortex and hippocampus93–95. It has been well established that CB1R expression highly concentrates on presynaptic terminals, where it mediates retrograde signaling of eCBs and functionally suppresses presynaptic GABA release69,96,97.
To investigate CB1R expression in TRN GABAergic neurons, we further performed FISH to label cnr1 mRNA and immunofluorescence staining to label GAD67 protein. We confirmed CB1R expression in TRN GABAergic neurons, which is consistent with results from in situ hybridization experiments conducted by the Allen Institute for Brain Science. Additionally, application of AM251, a CB1R antagonist, attenuated NaSal-induced GBO, hyperacusis, and anxiety-like behaviors. Although NaSal or AM251 may also impact local inhibitory circuits in the AC, PFC, or other anxiety-related regions, we selectively knocked out CB1Rs in TRN GABAergic neurons and found that NaSal injection no longer induced significant GBO enhancement and anxiety-like behaviors. This suggests that CB1R within the TRN is a key target involved in NaSal-induced aberrant behaviors.
Taken together, we propose a possible molecular mechanism underlying NaSal-induced hyperacusis and anxiety-like behaviors: NaSal increases the levels of eCBs by inhibiting the activity of COX-2, which mainly acts on the CB1Rs of GABAergic neurons in the TRN. The activation of eCBs/CB1Rs pathway suppresses presynaptic GABA release, leading to the disinhibition of neurons in the MGB and MD, the enhancement of GBO in the AC and PFC, and hyperacusis and anxiety-like behaviors in mice.
A study conducted intracranial recordings of the AC (i.e., electrocorticography) in a patient with severe tinnitus for 14 years, revealing an increase in gamma and theta activity in one of the eight implanted electrodes98. Although there are no invasive recordings in the human MGB currently, it is reasonable to believe that the MGB also plays a critical role in tinnitus pathology given its crucial role in integrating and processing multimodal upstream and downstream signals99. On the other hand, several clinical studies have reported exaggerated PFC and MD responses in patients with anxiety disorders100–102. The comorbidity of anxiety and tinnitus is common in clinical practice, but it remains uncertain whether anxiety causes tinnitus or if tinnitus causes anxiety. The results of our study suggest that anxiety and tinnitus may occur simultaneously due to the involvement of the TRN, which regulates both the auditory thalamo-cortical pathway associated with tinnitus and the limbic thalamo-cortical pathway associated with anxiety. Therefore, a definitive causal relationship between tinnitus and anxiety may not exist; instead, their comorbidity appears to stem from TRN dysfunction. Due to its deep location and small size, limited clinical data is available regarding research on the TRN. However, some researchers speculate that the TRN plays a crucial role in regulating functions such as emotions, higher order cognitive and sleep41,103,104. Acute cannabinoid overdose can cause feelings of panic and anxiety, hallucinations, and psychosis105. This indicates that the activation of CB1R can effectively decrease the release of inhibitory neurotransmitter GABA from neurons, leading to the hyperactivation of excitatory neurons. Consequently, the modulation of CB1R on the inhibitory function of TRN neurons may play a significant role in neuropsychiatric disorders such as tinnitus and anxiety.
We found the involvement of MGB-AC and MD-PFC neural hyperactivation in NaSal-induced auditory and emotional abnormalities, suggesting that thalamo-cortical hyperexcitation may underlie the potential neural mechanism for the comorbidity of tinnitus and anxiety disorders. Moreover, our study underscored the pivotal regulatory role of the TRN in the thalamo-cortical pathway and provided insights into a possible molecular mechanism involving eCBs/CB1Rs-mediated GABAergic presynaptic transmission. These findings point toward a novel direction for targeted therapy in managing comorbid tinnitus and anxiety disorders by enhancing the regulatory function of TRN.
Several limitations exist in our study. First, we did not directly assess tinnitus in the NaSal model mice because there is currently a lack of reliable behavioral or electrophysiological experimental methods to detect tinnitus in mice. Although the gap-prepulse inhibition (gap-PPI) experiment is used as a potential tool for assessing tinnitus in animals and humans, its results exhibit significant variability and cannot yet serve as a definitive criterion for tinnitus detection. Several studies have also reported abnormalities in gap-PPI in NaSal-treated mice106,107. It is worth noting that previous clinical research has frequently observed concurrent occurrences of tinnitus and hyperacusis108,109. Given that the detection of hyperacusis is relatively straightforward and reliable, in our study we used the ASR/PPI experiment to assess hyperacusis in NaSal-treated mice, which to some extent may indicate the possibility of tinnitus in this model. However, whether and to what extent NaSal induces tinnitus in mice requires future application of more reliable detection methods for confirmation. Secondly, our current findings show a correlation between hyperacusis and anxiety-like behaviors in NaSal-treated mice, but do not establish a direct causal relationship between them. Moreover, anxiety and tinnitus may co-occur due to some shared neuropathological mechanisms. This issue warrants further investigation for clarification in future research. Finally, we employed a 1 s ON-1s OFF stimulation pattern during the TRN activation experiment to reduce the risk of photodamage. However, this intermittent stimulation protocol may have only partially restored TRN neuronal activity, failing to reach its physiological baseline. Therefore, mice still exhibited some degree of anxiety, which does not fully prove the role of TRN in regulating auditory hypersensitivity and anxiety.
Methods
Animals and drugs treatment
All animal experimental protocols were approved by the Animal Ethics Committee of China Medical University in accordance with Institutional Animal Care and Use Committee guidelines for animal research. We have complied with all relevant ethical regulations for animal use. C57BL/6 mice (Vital River Laboratory, Beijing, China), FosCreER mice (B6.129(Cg)-Fostm1.1(cre/ERT2)Luo/J, #021882), Ai14 mice (B6.Cg-Gt(ROSA) 26Sortm14(CAG-tdTomato)Hze/J, #007914), GAD2-cre mice (Gad2tm2(cre)Zjh/J, #010802) and CB1f mice (B6.129P2(Cg)-Cnr1tm1.2Ltz/J, #036107) (Jackson Laboratory, ME, USA) were used. The FosTRAP mice were obtained from crossing female Ai14 mice with male FosCreER mice. All nomenclature and abbreviations for genotype combinations of transgenetic mice used in this study were listed in Supplementary Table 1.
All experiments were conducted using male mice aged 6 to 8 weeks, with a weight range of 20 to 25 g. Mice were housed in standard animal cages with sufficient food and water individually under controlled conditions (12 h light/12 h dark schedule, 22 ± 1°C, 30–50% humidity). All of the surgeries were performed under anesthesia to minimize the pain of animals.
For NaSal treatment, mice received an intraperitoneal (i.p.) injection of 300 mg/kg NaSal (HY-B0167A, MedChemExpress, NJ, USA) freshly diluted in 0.9% saline or an equal volume of saline (vehicle). For CB1 receptor antagonist AM251 treatment, AM251 (HY-15443, MCE) was first dissolved in 10% DMSO and then in 0.9% saline for i.p. administration at a final dose of 20 mg/kg.
Behavioral test
ASR/PPI test
ASR/PPI tests were conducted on the free-moving mice in standard startle chambers. One block for PPI testing consists of 6 different sound stimuli with 10 repetitions: 1) pulse-alone: 40 ms white noise burst (120 dB); 2–6) prepulse-pulse: 20 ms white noise burst (80, 85, 90, 95, or 100 dB) + 100 ms inter-stimuli interval (ISI) + 40 ms white noise burst (120 dB). Trails were presented in randomized order, with 25–30 s randomized inter-trial interval.
OFT
The OFT was performed in an open field apparatus (54 × 40 × 30 cm) constructed of non-transparent white boards for well position tracking. Before testing, each mouse underwent a 5-min habituation in the procedure room. Then the mouse was placed into the center of the arena, and was allowed to explore for 30 min. Activity of the mouse was monitored via an overhead camera (C270 HD Webcam, Logitech). The open field apparatus was cleaned with 70% ethanol between each mouse.
Home cage spontaneous activity
Mice were observed in their home cages by video recording for 30 min. The testing took place 3 h after the beginning of the dark cycle.
O-maze
The elevated O-maze was a modification of the elevated plus maze. The elevated circular platform (100 cm off the ground, 50 cm in diameter) had two enclosed arms opposite each other (10 cm wide with 12 cm high walls) and two open arms (10 cm wide). Briefly, each mouse was gently lowered by its tail into the open arena of the maze and given 9 min to explore. For the O-maze involving optogenetic manipulation of TRN, the exploration period lasted 12 min. Because the simulations in the TRN involved continuous optogenetic activation, the exploration period was split into six 2-min sections (OFF-ON-OFF-ON-OFF-ON).
For O-maze with MGB-AC and MD-PFC optogenetic manipulation, the exploration duration was 9 min, and for O-maze with TRN optogenetic manipulation, the exploration duration was 12 min. The whole circular platform was cleaned with 70% ethanol and wiped with paper towels between each animal.
Fos reporter activation
4-OHT was dissolved in ethanol to 20 mg/mL by shaking at 37 °C for 15 min, then aliquoted and stored at −20 °C for up to a month. Before use, 4-OHT was re-dissolved in ethanol by shaking at 37 °C for 15 min. Corn oil (HY-Y1888, MCE) was added to give a final concentration of 5 mg/mL, and the ethanol was evaporated by vacuum under centrifugation.
FosTRAP mice were given 7 days for the habituation of daily handling and i.p. injection. On day 8, the mice received an i.p. injection of 4-Hydroxytamoxifen (4-OHT, HY-16950, MCE 50 mg/kg)110,111, and another injection of vehicle/NaSal 30 min later. Mice were kept in their home cage for 72 h and then euthanized on day 11 for perfusion and immunohistochemistry analysis.
Tissue processing and immunohistochemistry
Mice were anesthetized and transcardially perfused with PBS and 4% PFA. Brains were dissected, postfixed in 4% PFA, and dehydrated in gradient sucrose. Embedded in Tissue-Tek O.C.T compound (Sakura Finetek, CA, USA), frozen brains were cut on a cryostat (Leica CM 1900, Leica Biosystems, Nussloch, Germany) into 15 μm-thick sections.
For immunostaining, sections were incubated in normal goat serum and primary antibodies (Anti-NeuN, 1:500, ab177487, Abcam, MA, USA; GAD67/Gad1 antibody, 1:300, A2938, ABclonal, Wuhan, China) overnight at 4 °C, followed by the appropriate fluorescent secondary antibody (1:300, Proteintech, Wuhan, China).
Combined FISH for Cnr1 and GAD67/Gad1 with immunostaining was conducted. The FAM-labeled Cnr1 probe (GenePharma, Shanghai, China) was used to detect Cnr1 in GAD67/Gad1-labeled cells. Briefly, FISH was performed in the frozen sections by using FISH kit (GenePharma) according to the manufacturer’s protocol. The slides were then washed and blocked for the immunostaining, and mounted in an anti-fade reagent with DAPI (SL1841, Coolaber, Beijing, China). All abbreviations of gene names and protein/receptor names in this study were listed in Supplementary Table 2.
Tissue sections were observed using a fluorescence microscope (BX53, Olympus, Tokyo, Japan). Cell counts for Fos and NeuN were obtained with IMARIS software (Bitplane, Zürich, Switzerland). The borders of regions were defined manually according to the Franklin and Paxinos mouse brain atlas112. All imaging and analyses were conducted in a blinded manner, without knowledge of the experimental conditions.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Supplementary information
Supplementary information
The online version contains supplementary material available at 10.1038/s42003-024-07040-5.
Acknowledgements
The study was funded by the Chinese National Key Technology R&D Program (2021YFC2501303 to P.T.Y.), the Joint Funds of the National Natural Science Foundation of China (U22A20309 to P.T.Y.), the Department of Science and Technology of Liaoning Province (2021JH1/10400049 to L.Q.), the ‘Xingliao Talent Plan’ of Liaoning Province (XLYC2002094 to L.Q.), National Natural Science Foundation of China (Youth Fund, No.82301533 to X.J.W.).
Peer review
Peer review information
Communications Biology thanks Shaowen Bao and Richardson Leao for their contribution to the peer review of this work. Primary Handling Editor: Benjamin Bessieres.
Data availability
The data that support the findings of this study are available from Supplementary Data 1.
Code availability
The custom codes used for analyzing data are available at https://github.com/JingyuChen96/FiberRecordingAndBehavioralAnalysis.
Competing interests
The authors declare no competing interests.