L-type calcium channel modulation reveals the relationship between neuronal synchrony and hyperactivity in the inferior colliculus following noise-induced hearing loss
Selin Yalcinoglu, Rod D. Braun, Ammaar Wattoo, Aaron K. Apawu, Rasheed Alrayashi, Avril Genene Holt
TL;DR
This study explores how blocking L-type calcium channels affects hearing and brain activity in rats after noise exposure.
Contribution
The study reveals the role of L-type calcium channels in neuronal synchrony but not hyperactivity in the inferior colliculus after noise exposure.
Findings
Verapamil prevented noise-induced decreases in auditory brainstem response amplitudes.
LTCCs are critical for neuronal synchrony in the inferior colliculus.
LTCC antagonism did not prevent noise-induced hyperactivity in the inferior colliculus.
Abstract
Previous studies have established the protective effects of calcium channel inhibition on the peripheral auditory system in response to noise exposure. While these studies implicate L-type calcium channels (LTCCs) in noise-generated dysfunction in the auditory periphery, contributions of LTCCs to noise-induced central dysfunction remains unclear. To begin to elucidate the roles of LTCCs in hearing, peripheral and central auditory function were assessed longitudinally after LTCC inhibition. Neuronal synchrony and activity were assessed by analyzing wave I (peripheral) and wave V (central) auditory brainstem responses (ABRs). Just prior to a noise exposure resulting in a temporary shift in hearing thresholds, rats were administered verapamil (LTCC blocker) or saline. Verapamil administration prevented the noise-induced decrease in ABR wave I and V amplitudes. Interestingly, when non-noise…
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Figure 9- —Department of Veterans Affairs
- —National Institutes of Health
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Taxonomy
TopicsHearing, Cochlea, Tinnitus, Genetics · Vestibular and auditory disorders · Transcranial Magnetic Stimulation Studies
Introduction
Hearing loss affects approximately 48 million Americans, with more than half having lasting damage due to excessive noise exposure^1^. Hearing loss can impact quality of life, cognition, and mental health with increased suicidal ideations^2^. Individuals whose occupational and recreational activities involve noisy environments are at higher risk for permanent or temporary hearing impairment (e.g., factory workers, military personnel, dental hygienists, musicians, or athletes)^1,3^.
Regardless of the severity of the noise-induced hearing loss (NIHL), there can be sustained changes in peripheral and central auditory-related function. Additionally, exposure to loud noise has been associated with synaptopathy (a loss of synaptic connection between inner hair cells and the auditory nerve in the periphery), and increased neuronal activity in central auditory brain regions, such as the inferior colliculus^4–6^.
Neuronal activity throughout the auditory pathway is primarily regulated by free calcium entering neurons via voltage-gated calcium channels, which trigger neurotransmitter release^7–9^. The L-type calcium channel family consists of four distinct subtypes (Cav1.1-Cav1.4), with Cav1.2 and Cav1.3 channels playing critical but distinct roles in auditory processing^10–13^. Cav1.3 channels are essential for synaptic transmission at inner hair cell (IHC) ribbon synapses in the cochlea^14^. These channels cluster at the presynaptic active zones of IHCs, where they mediate calcium influx that triggers glutamate release onto auditory nerve fibers^11–13,15,16^. In contrast, Cav1.2 channels are widely distributed in postsynaptic neurons throughout the brain, including the inferior colliculus, where they regulate synaptic plasticity and gene transcription^10,17–19^.
The afferent auditory pathway exhibits a distinct calcium channel subtype organization. At the peripheral level, synaptic transmission relies predominantly on Cav1.3 channels at IHC-auditory nerve synapses, which show relatively low sensitivity to verapamil and other phenylalkylamine calcium channel blockers^11–13,15,20^. Further upstream in the central auditory pathway, synaptic transmission depends primarily on P/Q-type (Cav2.1) channels, which are insensitive to verapamil^21–23^. These Cav2.1 channels are the predominant calcium channel subtype at auditory brainstem synapses, where they support the high-frequency, reliable synaptic transmission required for temporal coding of acoustic information^21–24^.
Verapamil, a phenylalkylamine L-type calcium channel antagonist, exhibits differential pharmacological selectivity between calcium channel subtypes^20,25–27^. The drug demonstrates markedly higher affinity for Cav1.2 channels compared to Cav1.3 channels, with studies showing 5–tenfold differences in inhibitory potency^20,25,26^. This selectivity profile arises from structural differences in the drug binding pockets between the two channel subtypes. The verapamil-sensitive Cav1.2 channels are present in almost all neurons (predominantly postsynaptic) and are crucially involved in activity-dependent synaptic plasticity and calcium-dependent gene transcription^10,19,27^.
The effects of increased calcium accumulation due to excessive neuronal activity can range from ototoxicity and cell death to increased spontaneous neuronal activity and have been linked to multiple auditory-related disorders^28–32^. Thus, regulation of calcium channel function might represent a potential therapeutic approach to protect from both peripheral and central contributions to NIHL. Previous studies in rodents have used various calcium channel antagonists to reduce the impact of noise-induced permanent threshold shifts in the periphery with some success^18,33–36^. However, there remains a significant gap in knowledge regarding the effects of calcium channel inhibition on noise-induced synaptic function centrally.
Verapamil, a non-dihydropyridine L-type calcium channel antagonist, has few side effects and readily crosses the blood–brain barrier to target calcium channels centrally.Voltage-gated calcium channel function and calcium mobilization are crucial for normal auditory transmission^14,37–39^. Since noise exposure disrupts spontaneous neuronal activity^17,20,30^, understanding the balance between hyperactivity and synchrony provides insight into noise-related neural manifestations.
Neuronal synchrony refers to the precise temporal coordination of compound action potentials (firing) across a population of neurons in response to auditory stimuli^37^. In contrast, hyperactivity refers to abnormally increased spontaneous or evoked neuronal firing, which can emerge after noise-induced trauma and may occur independently of synchrony^5,40–42^. Neuronal synchrony is crucial for the accurate encoding and transmission of auditory information, particularly for preserving temporal features of complex sounds^16,21,24^. Synchronization is influenced by various factors, including the properties and distribution of different voltage-gated calcium channels, which contribute to both the initiation and regulation of synchronous activity in the auditory pathway.
Therefore, we hypothesize that inhibition of L-type calcium channels will provide protection from noise-induced trauma for both peripheral and central auditory transmission through modulation of the dynamic interplay between synchrony (generated as the result of auditory nerve signaling) and neuronal activity (hypo- vs hyper-activity). The differential sensitivity of Cav1.2 versus Cav1.3 channels to verapamil, combined with their distinct anatomical distributions, predicts that calcium channel inhibition will have contrasting effects on peripheral versus central auditory function.
Materials and methods
Subjects and design
Twenty-five young adult male Sprague–Dawley rats (Charles River Laboratory, New Jersey) were randomly divided into four treatment groups. The treatment groups were no noise exposure with saline (n = 6), noise exposure with saline (n = 7), no noise exposure with verapamil (n = 7), and noise exposure with verapamil (n = 7). Approximately 20 min prior to the noise exposure, either verapamil (30 mg/kg) or saline (27 ml/kg, 0.9%) solution was administered intraperitoneally. The noise groups were exposed to a 16 kHz, 106 dB SPL tone for one hour, while the no noise control groups were maintained in ambient noise conditions (60 dB) for an equal amount of time.
All rats were individually housed and maintained at 24 ± 1° C with a 12-h light/dark cycle (lights on at 7 AM). Standard housing conditions with free access to normal rat chow and tap water were provided at Wayne State University’s (WSU) AAALAC-accredited animal facility. Animals were treated in accordance with the Animal Welfare Act. In addition, the DHHS “Guide for the Care and Use of Laboratory Animals” was followed. All procedures were approved by the WSU Institutional Animal Care and Use Committee in accordance with NIH (D16-00,198). This study is reported in accordance with ARRIVE guidelines.
Noise exposure
Just prior to noise exposure, a single ear of each animal was provided protection by filling the ear canal with a silicone elastomer for sound attenuation during the exposure (Kwik Seal, World Precision Instruments, Sarasota, FL). Awake and freely moving animals were exposed to a 16 kHz tone, 106 dB SPL with a sound pressure level (SPL) of 106 dB generated using software (Daqarta v 10.3) and delivered by overhead speakers for one hour. The silicone plug was removed immediately following the noise exposure. Animals in the no noise group had one ear plugged for one hour but were not exposed to loud noise.
Auditory brainstem responses (ABRs)
ABR analysis evaluated both the amplitudes (waves I and V) and hearing thresholds. Animals were anesthetized with an intramuscular injection of ketamine (75 mg/kg) and xylazine (8 mg/kg). To record the neurological response, subdermal electrodes were placed below the test ear (reference), below the contralateral ear (ground), and at the vertex (active). The assessment was performed at two different frequencies (12 kHz and 20 kHz) at incremental intensities (between 30–85 dB) at three time points (same day, one day, and five days after treatment; Fig. 1). At each intensity amplitudes (μV) and latencies (ms) were calculated. Amplitudes were calculated by subtracting the negative peak from the corresponding positive peak of the response, and latencies were recorded at only the positive peak for each wave (I and V). Amplitudes were assessed first at early (ET, day 0 and 1) and late (LT, day 5) time points and additionally at 0, 1, 5 days after treatment and exposure. Latencies were assessed at only ET and LT points. Hearing thresholds were measured at 12 and 20 kHz, with decreasing increments beginning at 80 dB SPL, until a response was no longer elicited. The lowest intensity that elicited a response was recorded as the threshold.Fig. 1. Experimental timeline. Following treatment (verapamil, V or saline, S) and noise exposure, each rat had ABR and ASR tests performed at three different time points: same day after treatment (day 0), one day after treatment (+ 1 day), and five days after treatment (+ 5 days).
Gap inhibition of Acoustic Startle Reflex (giASR)
giASR testing was performed at six frequencies (4, 8, 12, 16, 20 and 24 kHz) and two intensities (45- and 60-dB SPL). Testing was performed at three time points (same day, one day, and five days after treatment, Fig. 1). The gap inhibition of ASR paradigm was composed of three types of trials (Fig. 2): a silent period followed by a 20 ms startle sound (120 dB broadband noise; Fig. 2A, “sound-off startle” or “SOS” trial), a background tone immediately followed by a startle sound (Fig. 2B, “no gap” or “NG” trial), and a background tone interrupted by 50 ms of silence before reintroduction of the tone for 50 ms before the startle sound (Fig. 2C, “gap” or “G” trial) . In each trial F_max_, the maximum Newton force exerted in response to the startle sound, was recorded. Following the testing, the F_max_ in gap trial to F_max_ in sound-off startle trial (G/SOS) ratio was analyzed.Fig. 2ASR testing paradigm. Gap detection was composed of three types of trials: a silent period followed by a startle sound (solid black bar), i.e., “sound-off startle” or “SOS” trial (A), a background tone immediately followed by a startle sound, i.e., “no gap” or “NG” trial (B), and a background tone interrupted by a 50 ms silent period before reintroduction of the tone for 50 ms before the startle sound, i.e., “gap” or “G” trial (C). In each trial, Fmax (indicated by the arrow), the maximum force exerted during the startle response, was recorded.
Statistical analysis
All data were analyzed using StatView Version 5.0.1.0 (SAS Institute Inc.) and GraphPad Prism version 10.4.1 for MAC (Boston, MA USA). The data points were assessed using Kolmogorov–Smirnov test and was determined to be normally distributed. Group differences were analyzed by analysis of variance (ANOVA) and Tukey/Kramer post-hoc comparison. Significance of a linear regression was determined using a t-test. To determine if the slope of a line was significantly different from 1, an extra sum-of-squares F test was used. P values less than 0.05 were considered significant. All data are expressed as mean ± standard error of the mean.
Results
Verapamil does not impact hearing thresholds in normal or noise exposed animals
To assess whether L-type calcium channel inhibition affects baseline auditory sensitivity, hearing thresholds were measured at 12 and 20 kHz across all treatment groups at multiple time points following verapamil administration and noise exposure.
Same day of treatment
On the same day of treatment (Fig. 3A), a significant threshold shift was observed in the noise saline group compared to no noise saline and no noise verapamil groups at 12 kHz (26 dB SPL and 31 dB SPL, respectively, p < 0.05) and at 20 kHz (29 dB SPL, p < 0.05). The noise verapamil group had a significant threshold shift compared to no noise saline and no noise verapamil at 12 kHz (28 dB SPL and 30 dB SPL, respectively, p < 0.05) and at 20 kHz (33 dB SPL, p < 0.05). On average, the ear protected by the silicone elastomer produced a 27 dB attenuation of the threshold shift caused by noise exposure and was not significantly different from the control, no noise saline group.Fig. 3ABR thresholds at 12 and 20 kHz. Three different time points (A) same day of treatment, (B) one day after treatment, and (C) five days after treatment, are shown. Hearing thresholds of each treatment group (no noise saline, no noise verapamil, noise saline and noise verapamil) are shown at 12 kHz (hexagons) and 20 kHz (plus signs). Groups were compared using ANOVA with a post-hoc Tukey–Kramer test, and the error bars indicate the standard error of mean. * Asterisks denote significance at p < 0.05.
One day after treatment
One day after treatment, thresholds were still elevated in noise groups both at 12 and 20 kHz (Fig. 3B). A significant threshold shift was observed in the noise saline group compared to no noise saline and no noise verapamil groups at 12 kHz (13 dB SPL, p < 0.05) and at 20 kHz (21 dB SPL and 19 dB SPL, respectively, p < 0.05). The noise verapamil group had a significant threshold shift compared to no noise saline and no noise verapamil at 12 kHz (13 dB SPL, p < 0.05) and at 20 kHz (16 dB SPL and 15 dB SPL, respectively, p < 0.05).
Five days after treatment
Five days after noise exposure (Fig. 3C) hearing thresholds of both the noise groups returned to baseline with no significant difference compared to no noise groups.
Verapamil prevents early effects of noise on Wave I input/output (I/O) function
To determine whether L-type calcium channel inhibition protects peripheral auditory nerve fibers from noise-induced dys-synchrony, ABR Wave I amplitudes and latencies were analyzed as indicators of auditory nerve function and inner hair cell synaptic integrity.
I/O Function
For the 0 time point (day of noise exposure) there was significant decrease in the NS group amplitude compared to NNS and NNV groups at both 12 and 20 kHz (p ≤ 0.0001, Fig. 4A-B). Interestingly, there was also a significant decrease in the NS group amplitude compared to NV at 20 kHz (p = 0.0121, Fig. 4A). Additionally, the NV group is not significantly different compared to either the NNS and NNV groups at 12 or 20 kHz (Fig. 4A-B). By the 1 day time point, there were no significant differences amongst groups. Overall, at the 0 time point following noise exposure, there was a rightward shift of amplitude responses that was prevented by verapamil administration at both 12 and 20 kHz (Fig. 4A-B).Fig. 4ABR wave I amplitude and latency at 12 and 20 kHz. Wave I input/output function amplitude (A-B) and latency (C-D) across 5 intensity ranges are displayed. The wave I amplitudes and latencies were analyzed at three time points across four groups [no noise saline time points (circles: NNS 0—open, NNS 1—half filled, NNS 5—filled), no noise verapamil time points (squares: NNV 0—open, NNV 1—half filled, NNV 5—filled), noise saline time points (triangles: NS 0—open, NS 1—half filled, NS 5—filled), noise verapamil time points (closed diamonds: NV 0—open, NV 1—half filled, NV 5—filled), groups at 12 kHz (A, C) and 20 kHz (B, D). Groups were compared using an ANOVA with a Tukey/Kramer test used for post-hoc comparisons. Error bars indicate standard error of mean. Brackets with asterisks denote significance.
Latency
As expected, for each group at each time point Wave I latency at both 12 and 20 kHz (Fig. 4C-D) decreased as the intensity of the tone increased. However, noise disrupted this pattern and for the 5 day time point the NNS and NNV groups showed significantly smaller latencies (p = 0.0113) compared to the NV group at 12 kHz. No significant differences in latency were observed amongst groups at 20 kHz (Fig. 4C-D).
Verapamil exerts temporal effects on Wave V input/output (I/O) function after noise exposure
To evaluate whether verapamil changes noise-induced central auditory processing and inferior colliculus function, ABR Wave V amplitudes and latencies were examined as measures of I/O function and processing speed in the midbrain at different times following noise exposure.
I/O Function
At each time point for the NNS groups (0, 1, and 5 days) Wave V amplitude at both 12 and 20 kHz (Fig. 5B-C) increased as the intensity of the tone increased. Noise disrupted the normal I/O function at 12 kHz with the NNS group showing higher amplitudes when compared to the NS group on the day of noise exposure (p = 0.0001; Fig. 5A). Administration of verapamil reduced, but did not prevent, the effects of noise exposure (Fig. 5A). At 20 kHz the NNS 0 group compared to the NS 0 and NS 1 groups, were not significantly different. The NNV group at the 0 time point showed amplitudes of 0 uV regardless of the intensity tested at 12 kHz and only at the highest intensity tested for 20 kHz (p ≥ 0.080; Fig. 5A-B).Fig. 5ABR wave V amplitude and latency at 12 and 20 kHz. Wave V input/output function -amplitude (A-B) and latency (C-D) across 5 intensity ranges are displayed. The wave V amplitudes and latencies were analyzed at three time points across four groups [no noise saline time points (circles: NNS 0—open, NNS 1—half filled, NNS 5—filled ), no noise verapamil time points (squares: NNV 0—open, NNV 1—half filled, NNV 5—filled ), noise saline time points (triangles: NS 0—open, NS 1—half filled, NS 5—filled), noise verapamil time points (closed diamonds: NV 0—open, NV 1—half filled, NV 5—filled), groups at 12 kHz (A, C) and 20 kHz (B, D). Groups were compared using an ANOVA with a Tukey–Kramer test used for post-hoc comparisons. Error bars indicate standard error of mean. Brackets with asterisks denote significance.
Latency
Wave V latency at 12 kHz and 20 kHz, for the NNS groups (Fig. 5C-D) decreased as intensity of the tone increased. Noise disrupted this pattern at the two highest intensity ranges tested. At 12 kHz the NNS group at the 5 day time point remains faster than the NV group (p = 0.0001; Fig. 5C). Also, at 12 kHz the latency for the NNV group at the 5 day time point is significantly delayed compared to the NS group (p = 0.0001; Fig. 5C). For 20 kHz, NS at the 0 time point is significantly delayed compared to NS at the 5 day time point (p = 0.0038; Fig. 5D). The NS group at the 5 day time point is significantly faster compared to the NV group at this same time point (p = 0.0012; Fig. 5D). The NS is also significantly faster than the NNV group at the 5 day time point (p = 0.0001; Fig. 5D).
Verapamil prevents noise-induced synaptopathy at suprathreshold sound levels
To assess the effect of verapamil on noise-induced cochlear synaptopathy, Wave I amplitudes were compared following suprathreshold stimuli presented across time points in noise-exposed and non-exposed animals treated with verapamil.
12 kHz
On day zero of noise exposure there was a significant wave I amplitude reduction in the noise saline group compared to the no noise saline and the no noise verapamil groups (84% and 86% respectively, p < 0.05) at 12 kHz (Fig. 6A). The noise verapamil group was not significantly different when compared to the no noise saline and the no noise verapamil groups. One day after the noise exposure wave I amplitudes returned to normal for the noise saline group with no significant difference between the no noise groups (Fig. 6A). In addition, verapamil did not have any impact on wave I amplitude at later time points (Fig. 6A).Fig. 6ABR wave I amplitude for auditory brainstem responses at 12 and 20 kHz. Wave I amplitudes on day zero, and one and five days after treatment and noise exposure are displayed. The wave I amplitudes were analyzed for no noise saline (circles—NNS), no noise verapamil (squares -NNV), noise saline (triangles—NS) and noise verapamil (diamonds—NV) groups at 12 kHz (A) and 20 kHz (B). Groups were compared using ANOVA with a post-hoc Tukey–Kramer test, and the error bars indicate the standard error of mean. Brackets denote significance at p < 0.05. Shaded set of bars indicate the time point at which significant changes were observed.
20 kHz
On day zero of noise exposure there was a significant wave I amplitude reduction in the noise saline group compared to the no noise saline and the no noise verapamil groups (77% and 79% respectively, p < 0.05) at 20 kHz (Fig. 6B). The noise verapamil group was not significantly different when compared to the no noise saline and the no noise verapamil groups (Fig. 6B). One day after the noise exposure wave I amplitudes returned to normal for the noise saline group with no significant difference between the no noise groups (Fig. 6B). In addition, verapamil did not have any impact on wave I amplitude at later time points (Fig. 6B).
The impact of Verapamil on central synchronous activity is dependent on noise exposure status
To investigate how L-type calcium channel inhibition affects central auditory synchrony under normal conditions versus following noise trauma, ABR Wave V amplitudes were examined in both noise-exposed and non-exposed animals treated with verapamil.
12 kHz
On day zero of noise exposure, there is a wave V amplitude reduction in the noise saline group compared to no noise saline group at 12 kHz (p < 0.05, Fig. 7A). In addition, no noise verapamil group had a non-detectable wave V amplitude on day zero of treatment (p < 0.05, Figs. 7A, 8). However, on day zero, there is no wave V amplitude reduction in the noise verapamil group compared to the noise saline and no noise verapamil group (p < 0.05, Fig. 7A). One day after the noise exposure, wave V amplitudes returned to normal for no noise verapamil and noise saline groups with no significant differences among the other groups (Fig. 7A). In addition, verapamil did not have any impact on wave V amplitude at later time points (Fig. 7A).Fig. 7ABR wave V amplitude for auditory brainstem responses at 12 and 20 kHz. Wave V amplitudes on day zero, and one and five days after treatment and noise exposure are displayed. The wave I amplitudes were analyzed for no noise saline (circles—NNS), no noise verapamil (squares -NNV), noise saline (triangles—NS) and noise verapamil (diamonds—NV) groups at 12 kHz (A) and 20 kHz (B). Groups were compared using ANOVA with a post-hoc Tukey–Kramer test, and the error bars indicate the standard error of mean. Brackets denote significance at p < 0.05. Shaded set of bars indicate the time point at which significant changes were observed.Fig. 8. Auditory Brain Stem Response wave forms at 12 kHz at 80 dB SPL. Response to a tone pip at day 0 for no noise (saline or verapamil), and noise (saline or verapamil) groups are displayed. Wave I (auditory nerve), II (cochlear nucleus), III (superior olivary complex), IV (lateral lemniscus), V (inferior colliculus)^38,39^. are indicated on the no noise saline group. Wave I is indicated by a line and wave V is indicated by a gray bar. Waves that are present are marked by solid black arrowhead and waves that are absent are marked by a white arrowhead.
20 kHz
On day zero of noise exposure there is a wave V amplitude reduction in the noise saline group compared to no noise saline group at 20 kHz (p < 0.05, Fig. 7B). In addition, no noise verapamil group had a non-detectable wave V amplitude on day zero of treatment (p < 0.05, Fig. 7B). However, on day zero of noise exposure, there is no wave V amplitude reduction in the noise verapamil group compared to the noise saline and no noise verapamil group (p < 0.05, Fig. 7B). One day after the noise exposure wave V amplitudes returned to normal for no noise verapamil and noise saline groups with no significant differences among the other groups (Fig. 7B). In addition, verapamil did not have any impact on wave V amplitude at later time points (Fig. 7B).
Inhibition of the acoustic startle reflex occurs despite Verapamil-induced Wave V dys-synchrony
To further evaluate inferior colliculus activity during synchronous and dys-synchronous Wave V activity, gap inhibition of acoustic startle reflex (giASR) performance was assessed. Specifically, to determine the effects of Verapamil on startle inhibition performance, the degree of inhibition was evaluated with or without Verapamil in the noise and no noise groups.
G/SOS ASR Ratio at 45 dB
On day zero of treatment, the noise verapamil group showed a decrease in G/SOS ratio compared to the no noise saline (46% 12 kHz p < 0.05) and no noise verapamil (38%, at 12 kHz p < 0.05) group on day zero of treatment (Fig. 9A). By one day after treatment, the noise verapamil group showed a decrease in G/SOS ratio compared to the noise saline group at 20 kHz (47%, p < 0.05, Fig. 9B). Five days after treatment noise verapamil group showed an increase in G/SOS ratio compared to the no noise verapamil group at 20 kHz (33%, p < 0.05, Fig. 9B).Fig. 9. Gap to sound off startle ASR ratios at 12, and 20 at 45- and 60-dB SPL. The gap to sound off startle ASR ratios at three different time points, on day zero-, one- and five-day post treatment and noise exposure are displayed. The gap to sound off startle ASR ratios were analyzed for no noise saline (circles—NNS), no noise verapamil (squares—NNV), noise saline (triangles—NS) and noise verapamil (diamonds—NV) groups at 12 kHz (A, C), and 20 kHz (B, D) at 45 dB (A, B) and 60 dB (C, D). Groups were compared using ANOVA with a post-hoc Tukey–Kramer test, and the error bars indicate the standard error of mean. Brackets denote significance at p < 0.05.
G/SOS ASR Ratio at 60 dB
On day zero of treatment, the noise saline group showed a decrease in G/SOS ratio compared to the no noise saline group (65% and 54% at 12 and 20 kHz respectively p < 0.05 Fig. 9C-D) and no noise verapamil (50% at 20 p < 0.05, Fig. 9D). The noise verapamil group showed a decrease in G/SOS ratio compared to the no noise saline and no noise verapamil (70%, and 63% respectively, p < 0.05) groups on day zero of treatment at 12 kHz (Fig. 9C). By one day after treatment the noise verapamil group had significantly lower G/SOS ratios compared to no noise verapamil (63%, at 20 kHz, respectively, p < 0.05, Fig. 9D). Five days after treatment noise verapamil group showed deficits in G/SOS ratio compared to no noise saline group at 20 kHz (19%, p < 0.05, Fig. 9D).
There is an inverse correlation between ABR Wave V amplitude and giASR performance
To evaluate the relationship between ABR Wave I and Wave V amplitude (synchrony) with the degree of inhibition during giASR testing (inferior colliculus function), correlations were performed at 12 and 20 kHz .
For ABR Wave I, there was no significant correlation between amplitude and giASR ratios (i.e., G/SOS) in response to 12 or 20 kHz, 45 dB SPL carrier tones (R^2^ = 0.0029 and 0.0801 respectively; data not shown). A similar result was found when the carrier tone was 12 or 20 kHz, 60 dB SPL (R^2^ = 0.0094 and 0.1247 respectively, Fig. 10A-B). While there was no significant correlation between ABR Wave V amplitudes and giASRs elicited with 12 or 20 kHz, 45 dB carrier tones (R^2^ = 0.2074 and 0.1916 respectively, data not shown), there was a significant correlation between ABR Wave V amplitudes and G/SOS in response to 12 or 20 kHz, 60 dB carrier tones (R^2^ = 0.2270 and 0.2712 respectively, p ≤ 0.05 Fig. 10C-D).Fig. 10. Startle response ratio G/SOS – 60 dB SPL in relation to ABR Wave I and V amplitude at 12 and 20 kHz. Correlation of Wave I amplitude (A-B) and Wave V amplitude (C-D) and giASR (G/SOS) are displayed. The correlation was assessed at one day across groups no noise saline (circle—NNS), no noise verapamil (square—NNV), noise saline (triangle—NS) and noise verapamil (diamonds—NV). Significance of the slopes was determined using a regression t-test; p-values are shown.
Five days following noise exposure or Verapamil, central gain is enhanced
To assess changes in central auditory processing efficiency following noise exposure, the relationship between Wave I and Wave V amplitudes was analyzed to identify potential compensatory central gain mechanisms.
12 kHz
Five days following treatment and noise exposure the sound intensities evoked similar amplitudes for wave I and wave V in noise saline group compared to no noise saline group (the green and blue lines are not significantly different than the line of unity Fig. 11A). In addition, administration of verapamil prior to noise exposure did not change this effect, thus the sound intensities evoked similar amplitudes for wave I and wave V in the noise verapamil group compared to both the no noise saline and the noise saline group (the green and blue lines are not significantly different than the line of unity Fig. 11B and C respectively).Fig. 11. Tonal ABR wave amplitudes five days after treatment and noise exposure at 12 (A-C) and 20 kHz (D-F). Wave I (circles with green line) and wave V (squares with blue line) amplitudes for NS (A, D) and NV (B, C) vs. NNS. Additionally, NV vs NS wave I and wave V amplitudes are displayed (C, F). Red line represents the line of unity. Significance of the slopes was determined using a nonlinear fit from the line of unity; p-values are shown.
20 kHz
Five days following treatment and noise exposure, the sound intensities evoked similar amplitudes for wave I in the noise saline group compared to the no noise saline group (the green line is not significantly different than the line of unity Fig. 11D). However, the sound intensities evoked higher amplitudes for wave V in noise saline group compared to no noise saline group (the blue line is significantly above the line of unity Fig. 11D, p = 0.0360). The sound intensities evoked similar amplitudes for wave I in the noise verapamil group compared to the no noise saline and noise saline groups (the green line is not significantly different than the line of unity Fig. 11 E and F respectively). In addition, administration of verapamil prior to noise exposure resulted in sound intensities evoking lower amplitudes for wave V in noise verapamil group compared to noise saline groups (the blue line is significantly below the line of unity Fig. 11F, p = 0.0219). Thus, the sound intensities evoked similar amplitudes for wave V in the noise verapamil group compared to the no noise saline group (the blue line is not significantly different than the line of unity Fig. 11E).
Discussion
L-type calcium channels exhibit subtype-specific roles throughout the auditory pathway, with CaV1.3 channels governing transmitter release at inner-hair-cell ribbon synapses^11,12,14,30^., whereas CaV1.2 channels shape central synchrony in inferior-colliculus neurons^17,43^. The differential topographical distribution explains verapamil’s contrasting effects of peripheral protection versus central disruption, as the drug shows markedly different affinities for CaV1.3 (low affinity -micromolar) and CaV1.2 (high affinity -hundreds of micromolar) channels^20,25,44,45^. Pharmacological dissociation of synchronous and non-synchronous firing therefore provides a new window on the mechanisms that drive noise-induced auditory dysfunction^46^.
Peripheral auditory protection
Verapamil may attenuate noise-induced reduction of ABR Wave I (Figs. 4, 6) by limiting CaV1.3-dependent calcium influx at the inner-hair-cell synapse^13,31^. Although CaV1.3 channels are relatively insensitive^20,44,45^, partial blockade appears sufficient to curb calcium-driven excitotoxicity, preventing swelling of afferents and synaptic loss after acoustic trauma^4,31,32,35^. Consequently, verapamil may limit cochlear Ca^2^⁺ overload and microvascular damage after acoustic trauma^4,35,47^. While preserving auditory-nerve synchrony despite persisting threshold shift, underscoring that L-type channels safeguard synaptic integrity rather than auditory sensitivity^48^.
Central auditory effects
Systemic verapamil given to non-noise exposed animals produces an acute reduction in ABR Wave V amplitude (Figs. 5, 7, 8), consistent with transient suppression of inferior-colliculus gain that relies on tonically active L-type channels^43,49^. The high density of CaV1.2 currents in adult inferior-colliculus neurons^22,43^ accounts for this sensitivity, whereas rapid recovery of Wave V may reflect swift recruitment of P/Q-type CaV2.1 channels (insensitive to verapamil) that dominate mature auditory brainstem synapses^23,50^. Administered before noise exposure, verapamil preserves Wave V amplitude (Figs. 5, 7, 8), indicating that early L-type calcium entry is a trigger for central dys-synchrony^30^. Yet verapamil interferes with Ca^2^⁺-dependent gain control and synchronous firing in inferior colliculus neurons^43,49^.
Dissociation of hyperactivity and synchrony
The most striking finding of our study is the clear dissociation between neuronal synchrony and hyperactivity in the inferior colliculus (Figs. 7, 9) following noise exposure and calcium channel inhibition. Multi-unit recordings reveal that inferior-colliculus hyperactivity persists even when synchronous ABR components are reduced^40,51^; lesion studies show that such hyperactivity can survive loss of dorsal-cochlear-nucleus drive^41,52^. Enhanced gap-inhibition of the acoustic startle reflex therefore co-exists with diminished Wave V amplitude (Figs. 7, 8, 9, 10, 11, 12), supporting prior evidence that hyperactivity and neural synchrony are mechanistically distinct phenomena. Specifically, noise trauma is known to produce concurrent increases in spontaneous neuronal firing rates and reduced synchronous activity in the auditory brainstem^42^, with timing-dependent neural plasticity and synchronization specifically disrupted independently of general neuronal activity^53^. The observed inverse correlation between Wave V amplitude and giASR performance (Figs. 10, 11) further supports this paradoxical state of increased activity co-occurring with decreased synchrony in inferior colliculus. The complex relationship between synchronous and non-synchronous neuronal activity demonstrates that these processes can be pharmacologically dissociated, providing important insights into the mechanisms underlying noise-induced auditory dysfunction^30,46^.Fig. 12. Acoustic startle reflex and gap inhibition of acoustic startle pathway. The startle reflex involves the activation of the caudal pontine reticular nucleus and subsequent activation of motor neurons (A). Gap inhibition of the acoustic startle reflex (giASR) engages the inferior colliculus (B). Increase of inferior colliculus activity increases the acetylcholine release from the pedunculopontine tegmental nucleus to the caudal pontine reticular nucleus and a subsequent decrease in motor neuron activity. This ultimately leads to inhibition of the acoustic startle reflex.
Central gain and adaptive mechanisms
Selective modulation of L-type channels demonstrates that neuronal synchrony and hyperexcitability are separable dimensions of auditory coding^54,55^. Similar central-gain adaptations have been described after cochlear synaptopathy^46,56^ and work in the dorsal cochlear nucleus points to homeostatic re-balancing of excitation and inhibition as an underlying mechanism^57–59^. Because P/Q-type release machinery remains unaffected^23,50^, verapamil may curb maladaptive central gain while sparing essential temporal processing, highlighting the therapeutic promise of subtype-targeted calcium-channel modulators^19,60^.
The prevention of noise-induced central gain by verapamil administration (Fig. 7), reveals another important aspect of calcium channel function in auditory processing. Under normal circumstances, noise exposure triggers compensatory increases in central auditory responsiveness that may contribute to hyperacusis and other auditory processing disorders^61,62^. Verapamil’s ability to dampen this maladaptive central gain while preserving peripheral function suggests that L-type calcium channels play a crucial role in the plastic changes that follow acoustic trauma^63,64^. This finding has important implications for understanding how the auditory system attempts to compensate for peripheral damage and how pharmaceutical interventions might be used to prevent harmful adaptations while preserving beneficial ones^65–67^.
Study limitations and future directions
Several important limitations must be acknowledged in interpreting these results. The small sample sizes, particularly for critical comparisons involving the no noise verapamil group, limit statistical power and the ability to generalize findings. The single-dose, acute administration protocol, while appropriate for demonstrating proof-of-concept, may not reflect optimal therapeutic dosing strategies. The relatively high verapamil dose employed, while within established experimental ranges^68^, may produce off-target effects that complicate interpretation of results^10,69^.
Future studies could employ dose–response protocols, larger sample sizes, and subtype-selective calcium channel antagonists to better characterize the specific contributions of CaV1.2 versus CaV1.3 channels to auditory function^70,71^. Additionally, direct measurement of the expression and distribution of alternative calcium receptors and calcium imaging studies would help validate the proposed compensatory mechanisms and their role in maintaining inferior colliculus hyperactivity during L-type channel blockade^15,72,73^.
Conclusions
This study demonstrates that L-type voltage-gated calcium channels differentially contribute to peripheral and central auditory function through subtype-specific mechanisms^21,24^. L-type calcium channel inhibition reveals the complex relationship between synchronous and non-synchronous neuronal activity in the auditory system, showing that these processes can be pharmacologically dissociated^16,74^. The findings suggest that calcium channel dysfunction may represent an underlying mechanism for disorders involving altered auditory synchrony^75,76^, while compensatory pathways may maintain hyperactivity states^77,78^. Understanding these relationships enhances our knowledge of noise-induced auditory dysfunction mechanisms and supports the development of targeted therapeutic approaches for auditory disorders involving disrupted calcium homeostasis^79^.
The reference list from the paper itself. Each links out to its DOI / PubMed record.