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. 2014 Oct 13;592(Pt 22):5065–5078. doi: 10.1113/jphysiol.2014.278572

Single unit hyperactivity and bursting in the auditory thalamus of awake rats directly correlates with behavioural evidence of tinnitus

Bopanna I Kalappa 1, Thomas J Brozoski 2, Jeremy G Turner 2,3, Donald M Caspary 1,2,✉
PMCID: PMC4259543  PMID: 25217380

Abstract

Tinnitus is an auditory percept without an environmental acoustic correlate. Contemporary tinnitus models hypothesize tinnitus to be a consequence of maladaptive plasticity-induced disturbance of excitation–inhibition homeostasis, possibly convergent on medial geniculate body (MGB, auditory thalamus) and related neuronal networks. The MGB is an obligate acoustic relay in a unique position to gate auditory signals to higher-order auditory and limbic centres. Tinnitus-related maladaptive plastic changes of MGB-related neuronal networks may affect the gating function of MGB and enhance gain in central auditory and non-auditory neuronal networks, resulting in tinnitus. The present study examined the discharge properties of MGB neurons in the sound-exposure gap inhibition animal model of tinnitus. MGB single unit responses were obtained from awake unexposed controls and sound-exposed adult rats with behavioural evidence of tinnitus. MGB units in animals with tinnitus exhibited enhanced spontaneous firing, altered burst properties and increased rate-level function slope when driven by broadband noise and tones at the unit's characteristic frequency. Elevated patterns of neuronal activity and altered bursting showed a significant positive correlation with animals’ tinnitus scores. Altered activity of MGB neurons revealed additional features of auditory system plasticity associated with tinnitus, which may provide a testable assay for future therapeutic and diagnostic development.

Introduction

Tinnitus is the perception of phantom sound that affects an estimated 5–15% of the general adult population (Axelsson & Ringdahl, 1989; Nondahl et al. 2002; Shargorodsky et al. 2010). Among war veterans, tinnitus has been the most prevalent service-related disability since 2008 (http://www.vba.va.gov/reports/abr/index.asp). The psychological sequellae of severe tinnitus include depression, anxiety, disturbed sleep patterns and suicidal tendencies that severely affect the tinnitus sufferer's quality of life (Cooper, 1994). Despite its high prevalence and debilitating effects, the pathophysiology of tinnitus is poorly understood because of its complexity and subjective nature. As a result, there is no generally accepted cure, palliative treatment or adequate prevention for this disorder.

Tinnitus aetiology includes otological, neurological, infectious and drug-related causes (Lockwood et al. 2002). Regardless of aetiology, tinnitus is generally initiated by some degree of cochlear/acoustic nerve damage, resulting in reduced auditory nerve input to the central auditory system (Lockwood et al. 2002; Roberts et al. 2010; Henry et al. 2014). Reduced peripheral input triggers a series of compensatory plastic changes in central auditory structures that lead to the neural correlates of tinnitus, including altered neural activity, altered neurosynchrony and sensory map reorganization, all of which have been hypothesized to underpin tinnitus (Roberts et al. 2010; Eggermont, 2013; Zhang, 2013). The observed alterations in neuronal activity and/or network properties have been attributed to plastic changes in neurotransmitter systems including glycinergic, GABAergic, glutamatergic and cholinergic systems (Wang et al. 2009; Roberts et al. 2010; Kaltenbach, 2011; Eggermont, 2013; Zhang, 2013) as well as intrinsic neuronal properties (Li et al. 2013).

While tinnitus-related neural changes have been studied in many auditory structures, the medial geniculate body (MGB, auditory thalamus) has received relatively little attention. The MGB is an obligatory structure of the auditory neuraxis involved in shaping the sensory code and gating the relative salience of sensory signals (Bartlett & Smith, 1999; Bartlett & Wang, 2007,; Winer et al. 2011). The MGB receives ascending lemniscal and extra-lemniscal inputs that then project to auditory cortical and limbic centres, as well as receiving descending inputs from reticular, limbic, auditory and non-auditory cortices (Winer & Larue, 1987; LeDoux et al. 1991; Bajo et al. 1995; Winer et al. 1999,; Lee & Winer, 2008). Given its unique position and role in the integration and processing of multimodal upstream and downstream signals, it is reasonable to hypothesize a key involvement of the MGB in tinnitus pathology (Leaver et al. 2011). Moreover, homeostatic plasticity following decreased peripheral input has been previously reported in visual (Krahe & Guido, 2011), auditory (Kamke et al. 2003; Su et al. 2012; van Gendt et al. 2012) and somatosensory (Krahe & Guido, 2011) thalamus. Furthermore, tinnitus models such as thalamocortical dysrhythmia (Llinas et al. 2005) and noise cancellation (Leaver et al. 2011) suggest that tinnitus-related compensatory plastic changes may occur in MGB-related networks involving auditory cortex and thalamic reticular nucleus (Llinas et al. 2005; Yu et al. 2009; Leaver et al. 2011). Plastic thalamocortical dysrhythmic changes may alter sensory gating and enhance gain at auditory and limbic centres leading to an auditory percept with an emotional component. Based on the aforementioned observations and models, we investigated potential changes in the discharge properties of MGB neurons in tinnitus using single unit recordings from awake unexposed controls and awake sound-exposed animals selected for behavioural evidence of tinnitus.

Methods

Animals

Fifteen young adult Long Evans rats (3–6 months) were utilized in this study. The animals were individually housed with ad libitum access to food and water. A reverse light/dark cycle was used to enhance the probability that daytime recordings would be obtained during the rats’ active period. Animal handling and care were in accordance with the Southern Illinois University-School of Medicine Lab Animal Care and Use Committee (LACUC) approved guidelines and protocols.

Sound exposure paradigm

Sound exposure was similar to that used in previous studies (Bauer et al. 1999; Turner et al. 2006). In short, at 3–4 months of age, rats were anaesthetized with ketamine HCl (105 mg kg−1, Aveco, Fort Dodge, IA, USA) and xylazine (7 mg kg−1, Lioyd Laboratories, Shenandoah, IA, USA). This regimen provided excellent areflexia during the first threshold test and the 1.0 h sound exposure. A ketamine booster (0.1 ml) was administered prior to the second threshold test if there was a response to tail pinch or if pedal withdrawal was noted. Animals were subsequently observed until fully ambulatory. Sound-exposed rats were unilaterally exposed to a noise of a peak-calibrated level of 116 dB, centred on a 16 kHz octave band for 1 h using a Grason Stadler Noise Generator (Model 1724, Eden Prairie, MN, USA), shaped with a Krohn-Hite Filter (Model 3384, Brockton, MA, USA), and amplified with a Sony Stereo Power Amplifier (Model TA-N55ES, New York, NY, USA). Fostex horn tweeters (FT17H, Agoura Hills, CA, USA) fitted with a custom metallic cone funnel with a 5 cm long plastic tube (2 mm inner diameter) were used to present acoustic signals to one ear. This exposure leads to a unilateral temporary threshold shift in exposed rats as indicated by auditory brainstem response, which recovers to statistically normal levels over time (Wang et al. 2009). There were no observed threshold shifts in the unexposed ear 60 days following the exposure. Previous studies have found that unilateral hearing loss alone (simulated by plugging one ear, to produce a 22 dB threshold shift at 10 kHz) did not significantly affect gap detection (Turner et al. 2006).

Behavioural testing in rat model of tinnitus

Behavioural testing of tinnitus, 2 months following exposure, was conducted using startle reflex software and hardware, customized for this application (Kinder Scientific, Poway, CA, USA) as previously described (Turner et al. 2006). Briefly, background sounds in the startle chamber consisted of 60 dB SPL broadband noise (BBN) or 1/3 octave band pass filtered noise centered at 10, 12.5, 16, 20 and 25 kHz. Gap and pre-pulse inhibition (PPI) testing used background sounds presented through one speaker and startle stimuli presented through a second speaker located in the ceiling of the chamber. The floor of the animal chamber was attached to a piezoelectric transducer, which provided the measure of startle force (calibrated and measured in Newtons) applied to the floor. Gaps were always embedded 100 ms before startle stimuli, and were 50 ms in duration with a 1 ms rise/fall gate. Gaps preceding a startle stimulus by 100 ms produce a stable gap-induced inhibition of the startle reflex in rats (Ison, 1997; Turner et al. 2005). The gap PPI of acoustic startle (GPIAS) tinnitus test is based on the principle that animals with tinnitus should have a more difficult time processing silent gap cues as test signals because their tinnitus would degrade the quality of the silent gap cues, and that this deficit would be greatest when the background test sounds more closely matched the features of the animal's tinnitus (Turner et al. 2006). PPI using the same 1/3 octave band, 60 dB test signals was routinely measured serving as a control for hearing loss or sensory gating failures. If deficits in gap processing and/or PPI suggested that hearing loss was a contributor to the gap deficit, the animal was removed from the study.

Animal selection and standardization of tinnitus score

Animals were considered to have tinnitus if the gap–startle ratio of sound-exposed animals was above the upper limit of the 95% confidence interval (CI) of the mean gap–startle ratio of control animals for at least one background test frequency in the GPIAS paradigm. All sound-exposed animals used in this study showed GPIAS evidence of tinnitus at least at one frequency. If animals exhibited criterion-level gap–startle ratios at more than one test frequency then the frequency at which the gap–startle ratio deficit was the greatest was considered to be their tinnitus frequency. To determine whether a correlation exists between electrophysiological measures (e.g. spontaneous firing rates (SFRs), bursting) and animals’ gap–startle ratio, the raw gap–startle ratio at each test frequency for all the animals in this study was converted to a standardized score. Tinnitus was quantified relative to control (unexposed) GPIAS performance, using the formula z = (x – μ)/σ, where z is the standardized tinnitus score, x is the raw gap–startle ratio, and μ is the mean and σ the standard deviation of the control group. Once a z-score for every animal at each test frequency was established the highest z-score across test frequencies was chosen as the standardized tinnitus score for that animal (see Figs 2B, 3B, D, F, and 4B–E and G–J). All animals irrespective of experimental sound exposure were assigned the standardized tinnitus score, and results were compared on the basis of that score, i.e. higher z-scores indicating the greater likelihood of tinnitus. Experiments were conducted in a single blinded format with the experimenter collecting and analysing the electrophysiological data having no knowledge of tinnitus score or whether the animals were sound exposed or unexposed.

Figure 2. Characteristic frequencies (CFs) and thresholds of MGB units recorded from unexposed control animals and sound-exposed animals with behavioural evidence of tinnitus (tinnitus animals).

Figure 2

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A, scatter plot of individual MGB unit thresholds at their CF from control (blue) and tinnitus (red) rats. B, average threshold at CF of MGB units recorded from control (blue) and tinnitus (red) rats did not exhibit any significant difference (P = 0.234; unpaired t test, two-tailed). +, Mean firing threshold. Error bars represent SEM.

Figure 3. Spontaneous firing rates (SFRs) of MGB units recorded from unexposed control (control animals) and sound-exposed animals with behavioural evidence of tinnitus (tinnitus animals).

Figure 3

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A, average SFRs of MGB units recorded from tinnitus animals (9.09 ± 0.9423; red, six animals) were significantly elevated (P = 0.0005; unpaired t test, two-tailed) in comparison to controls (4.68 ± 0.48; blue, nine animals). +, Mean SFR. B, scatter plot of average SFR of MGB units recorded from individual animals (y-axis) Showing a significant correlation (y = 1.830x + 4.812, P = 0.004; Pearson correlation coefficient) with the animals’ tinnitus z-scores (x-axis). Blue and red squares represent control and sound-exposed animals, respectively. Error bars represent SEM.

Figure 4. Spontaneous bursting activity of MGB units recorded from unexposed control (control animals) and sound-exposed animals with behavioural evidence of tinnitus (tinnitus animals).

Figure 4

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A, C and E, MGB units recorded from tinnitus animals (red; six animals) exhibited significantly elevated, average bursts per minute (P = 0.0001) (A), average spikes in a burst (P = 0.009) (C) and average burst duration (P = 0.043) (E) compared to control rats (blue; nine animals). Unpaired t test was used to determine the statistical significance between the two groups. +, Mean of the test variable. B, D and F, scatter plots of average burst variables (y-axis) of MGB units from individual animals against that an animal's tinnitus z-score (x-axis). A significant correlation (y = 11.98x + 9.619, P = 0.001) was observed between average bursts per minute and animals’ tinnitus scores (B). However, average spikes in burst (P = 0.082) (D) and average burst duration (P = 0.237) (F) did not exhibit significant correlation with the animals’ tinnitus z-scores. Pearson correlation coefficient was used to determine statistical significance of the correlations. Blue and red squares represent control and sound-exposed animals, respectively. Error bars represent SEM.

Tetrode microdrives

The assembly and implantation of tetrodes and single unit recording conditions has been described in detail by Richardson et al. (2013). In short, each assembled VersaDrive4 tetrode drive (Neuralynx, Inc., Bozeman, MT, USA) contained four tetrodes clipped to a scaled length of ∼9 mm, the length required to advance completely through the MGB. Tetrode wires were 0.0007 μm in diameter made of platinum (10%) and iridium (90%) (California Fine Wire Company, Grover Beach, CA, USA). Once assembled, tetrodes were gold-electroplated to lower the impedance to within a narrow desired range of 1–1.2 MΩ sampled at 1 kHz (nanoZ, Neuralynx). An assembled VersaDrive4 tetrode drive weighed about ∼ 1.6 g with dimensions of 11 mm in width and 15 mm in height. Prior to implantation tetrodes were gas sterilized with ethylene oxide.

Pre-surgery animal handling

Animal handling began 1 week before surgery to ensure that the animal was comfortable with handling and mild restraint. During this period the rats were acclimatized, for a minimum of 5 days, to a modified experimental conditioning unit (restraining chamber) (ECU; Braintree Scientific, Braintree, MA, USA) using a food reward (fruit loops) and unrestricted access to water. On day 1, the rat was placed in the restraining chamber for ∼30 min. This was followed by a daily increment of 30 min to a final fifth day restraining time of ∼2.5 h. By the end of the acclimatization period the rats learned to sit quietly and still in the chamber for up to ∼2.5 h while having free access to water and food.

Chronic implantation surgery and maintenance

One day prior to surgery, rats were given acetaminophen (4.5 mg ml−1) via drinking water. For surgery, isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane;2.5-3%) (VetEquip, Inc., Pleasanton, CA, USA) was used to induce anaesthesia followed by i.m. administration of ketamine and xylazine (7 mg kg−1 xylazine; 105 mg kg−1 ketamine). Once anaesthetized, the head was shaved and the rat was transferred to a stereotaxic apparatus (David Kopf Instruments., Tujunga, CA, USA) where the head was stabilized in a standard position with jaw bars. The stereotaxic apparatus was fitted with a nose cone for continuous delivery of oxygen to maintain 95–100% blood saturation level. Isoflurane (1–2.5%) was delivered as required to maintain the anaesthetized state. A thermostatically controlled heating pad was placed underneath the rat and the temperature was monitored using a rectal thermometer probe (Harvard Apparatus, Holliston, MA, USA). O2 blood saturation level and heart rate were also constantly monitored (PulseSense Vet, Nonin Medical, Minneapolis, MN, USA). The shaved head was disinfected with alternating nolvasan and 70% alcohol. Before surgery rats were administered 2–3 ml s.c. sterile saline and ophthalmic ointment was applied to both eyes.

Under sterile surgical conditions, a skin incision was made over the skull and the skull surface was cleaned of remaining soft tissue. A 2.3 mm diameter burr hole was drilled over the left occipito-parietal cortex (–5.5 to 5.7 mm bregma and 3.5–3.8 mm of midline) to access the left MGB. To provide support screws and a ground screw, four additional holes were drilled (one in the posterior left frontal, one in the anterior left parietal, one in the right anterior parietal and one in the posterior right parietal bones). The tetrode ground wire was attached to the screw in the anterior parietal hole that made contact with the dura. The tetrode was advanced through the left posterior parietal hole to a depth of 4.5–5 mm placing the four tetrode tips just above dorsal MGB. Once positioned the electrode was secured to the skull using dental acrylic that encapsulated the anchor screws and the VersaDrive4 with the exception of the tetrode drive screws and connector located on top of the drive. After surgery, the rats were administered 2–3 ml s.c. sterile saline and triple antibiotic ointment was applied to the wound. The rats were then kept under observation and 100% oxygen was continued until they were ambulatory. Acetaminophen treatment (4.5 mg ml−1) was continued for 2 days after surgery while triple antibiotic application was continued for 4 days or until the wound healed.

Single unit recording

During data acquisition, the connector on the VersaDrive4 tetrode drive was coupled to an 18 pin (16 single wires, 2 ground wires) omnetics adaptor (Neuralynx) which in turn was connected to a unity gain 18 channel headstage and a pre-amplifier (2× gain, 0.15 kHz (high pass); 8 kHz (low pass); Plexon, Inc., Dallas, TX, USA). Using a multichannel acquisition processor and Sort Client (Plexon) 16 channels were digitized. Single units were identified using amplitude windowing and principal component analysis (Plexon). Unit data were recorded as time stamps and analysed using custom software (Auditory Neurophysiology Experiment Control Software – ANECS, Ken Hancock, Blue Hills Scientific, Boston, MA, USA). When unit data acquisition was complete, the tetrode was manually advanced in 250 μm increments. Single units within 125 μm depth of the previously recorded unit were discarded to avoid sampling the same unit twice. Animals from both control and tinnitus groups were treated in a similar manner as the experimenter was blinded to the groups. The experimenter took precautions during the recording session to ensure that the animal was not asleep. In addition, whenever there was an unexpected change in the firing rates of a single unit under investigation, data collection was paused and the booth door was opened to make sure the animal was not asleep. As noted above, animals were kept on a reverse day/night cycle so their active period was during the recording sessions. Once the tetrodes were advanced to their maximum advanceable limit, the rats were anaesthetized with ketamine and xylazine (as previously described) and current pulses (5 μA for 5 s) were passed through the tip of each tetrode wire to produce a small marker lesion. Following lesioning, rats were cardiac perfused with PBS (0.1 m, pH 7.4) followed by paraformaldehyde solution (4%). The brains were removed and placed in paraformaldehyde solution (1–2 h) and then transferred to sucrose solution (20%). Coronal sections (∼40 μm) were made using a cryostat (Leica CM 1850 – Leica Microsystems, Buffalo Grove, IL, USA), stained with thionin and analysed to determine the recording sites relative to the location of the tetrode tip. An example of an electrode track through the MGB from an implanted Long Evans rat is shown in Fig. 1.

Figure 1. Example of thionin-stained coronal section containing medial geniculate body (MGB).

Figure 1

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White dashed line represents the electrode track passing through dorsal (dMGB) and ventral MGB (vMGB) demarcated by dashed red lines. Scale bar = 500 μm. CA = Cornu Ammonis.

Acoustic stimuli and auditory testing

Acoustic signals were generated using a 16-bit D/A converter (TDT RX6, Tucker Davis Technologies (TDT) System III, Alachua, FL, USA), amplified (Yamaha P2500S, Buena Park, CA, USA) and transduced by a Fostex tweeter (model FT17H) placed 30 cm above the restraining chamber. Output of the tweeter was calibrated off-line using a ¼ inch microphone (model: 4938; Brüel & Kjær, Naerum, Denmark) placed in the restraining chamber at the approximate location of the rat's head as described previously (Caspary et al. 2005; Richardson et al. 2013). Calibration tables in dB sound pressure level (SPL) were then used to set programmable attenuators (TDT PA5) to achieve pure tone levels accurate to within 2 dB SPL for frequencies up to 45 kHz. Response maps were used to determine the characteristic frequency (CF) of sorted single units. Random tone-burst stimuli (50 ms duration, 4 ms rise/fall time, 2 Hz rate) were presented in 0.10 to 0.25 octave frequency steps (1–42 kHz) in 10 dB SPL steps (0–80 dB) to determine the response maps. Rate-level functions were generated using BBN or characteristic frequency tones (200 ms duration, 4 ms rise/fall time, 2 Hz rate) presented in random across trials fashion. Real time single unit activity was sampled at 100 kHz using ANECS (Ken Hancock, Blue Hills Scientific) and archived for off-line analysis.

Data and statistical analyses

Sorted single unit spontanteous activity was recorded for 5 min periods and stored as time stamps in Plexon files. Spike frequency and bursting were determined off line using Neuroexplorer.v. 4.097 (Nex Technologies, Inc., Dallas, TX, USA). Bursts were defined using time and frequency parameters similar to those previously described (Bauer et al. 2008): maximum inter-spike interval at burst start (500 ms), maximum interval to end burst (500 ms), minimum interval between bursts (500 ms), minimum burst duration (5 ms) and minimum number of spikes in a burst. Rate-level functions were analysed using ANECS. Independent t tests and Pearson correlation coefficients were used for statistical comparisons. A significance level of 0.05 was used for all statistical tests. All values are expressed as means ± SEM.

Results

Basic properties of MGB single unit responses

A total of 109 single units that responded to auditory stimuli were recorded from the MGB of awake unexposed controls (n = 64, nine animals) and sound-exposed Long Evans rats with behavioural evidence of tinnitus (n = 45, six animals). Each unit's CF and thresholds are plotted in Fig. 2A. There was no significant difference (P = 0.234; unpaired t test, two-tailed) between the mean threshold of MGB units recorded from unexposed control animals (30.16 ± 1.53 dB) and sound-exposed animals with tinnitus (33.04 ± 1.91 dB) (Fig. 2B). The discharge patterns of MGB units included ‘onset’ (transient responses only during stimulus onset followed by suppression of responses until stimulus offset), ‘offset’ (transient responses only after stimulus offset), ‘onset plus offset’ (transient responses during both stimulus onset and after stimulus offset), ‘sustained’ (responds by firing during the entire duration of stimulus) and ‘poor’ (weak variable firing patterns) response types. The distribution of unit response types from unexposed control animals and sound-exposed animals with tinnitus were comparable and is summarized in Table1.

Table 1.

Response types of MGB single units recorded from unexposed control and sound-exposed animals with behavioural evidence of tinnitus in response to broadband noise (BBN) and each unit's characteristic frequency (CF)

BBN
 CF
Type Unexposed controls Sound-exposed tinnitus Unexposed controls Sound-exposed tinnitus
Onset 32 (50.0%) 21 (46.7%) 31 (48.4%) 19 (42.2%)
Offset 13 (20.3%) 9 (20.0%) 15 (23.4%) 11 (24.4%)
Onset + Offset 7 (10.9%) 7 (15.6%) 9 (14.1%) 6 (13.3%)
Sustained 5 (7.8%) 2 (4.4%) 3 (4.7%) 4 (8.9%)
Poor 7 (10.9%) 6 (13.3%) 6 (9.3%) 5 (11.1%)
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Elevated SFRs of MGB units in tinnitus animals

The SFR of MGB units was recorded over a period of 5 min. While the mean SFR of units recorded from control animals (4.68 ± 0.48 spikes s−1; n = 63, nine animals) was comparable to previous reports (Richardson et al. 2013), the mean SFR recorded from tinnitus animals (9.09 ± 0.94 spikes s−1; n = 42, six animals) was significantly elevated (P = 0.0005; unpaired t test, two-tailed; Fig. 3A). A summary of SFRs from units in the lemniscal and non-lemniscal subdivisions of MGB from both groups are reported in Table2. Furthermore, when individual animal SFRs were plotted with their tinnitus z-score, a statistically significant positive correlation was obtained (r2 = 0.47; P = 0.004; Fig. 3B). Therefore, MGB units recorded from animals with higher z-scores, i.e. greater behavioural evidence of tinnitus, irrespective of sound exposure, were likely to show higher SFRs compared to animals with lower z-scores (Fig. 3B).

Table 2.

Summary of spontaneous firing rates (SFRs) and spontaneous bursting in unexposed control and sound-exposed animals with behavioural evidence of tinnitus from lemniscal and non-lemniscal subdivisions of medial geniculate body (MGB)

Unexposed controls
 Sound-exposed tinnitus
Non-lemniscal dMGB & mMGB (n = 22) Lemniscal division ventral (n = 42) Non-lemniscal dMGB & mMGB (n = 17) Lemniscal division ventral (n = 28)
SFRs (spikes s−1) 3.5 ± 0.7 5.6 ± 0.3 6.1 ± 0.9 (P < 0.05) 11.7 ± 1.2 (P < 0.0001)
Bursting (bursts min−1) 5.7 ± 2.9 8.6 ± 1.2 14.8 ± 3.3 (P < 0.05) 51.7 ± 8.1 (P ≤ 0.0001)
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Both subdivisions exhibited a significant increase in SFRs and spontaneous bursting in sound-exposed animals with behavioural evidence of tinnitus compared to control groups.

Elevated spontaneous bursting of MGB units in tinnitus animals

Neuronal burst firing is commonly defined as a rapid series of two or more spikes separated by a quiescent period (Grace & Bunney, 1984; Overton & Clark, 1997). In the present study, the spontaneous activity of MGB units was examined for bursting. Comparison of spontaneous MGB unit bursting between unexposed control animals (n = 63, nine animals) and tinnitus animals (n = 42, six animals) revealed a significant tinnitus-related increase in: (1) mean bursts per minute (9.20 ± 1.95, control vs. 37.06 ± 5.54, tinnitus; P = 0.0001; unpaired t test, two-tailed; Fig. 4A); (2) mean spikes in a burst (5.06 ± 0.14, control vs. 6.13 ± 0.37, tinnitus; P = 0.009; unpaired t test, two-tailed; Fig. 4C); and (3) mean burst duration (60 ± 0.00 ms, control, vs. 90 ± 10.0 ms, tinnitus; P = 0.043; unpaired t test, two-tailed; Fig. 4E). A summary of spontaneous bursting in the lemniscal and non-lemniscal subdivisions of MGB from both groups are reported in Table2. Furthermore, when mean unit burst parameters from individual animals were examined for positive correlations with animals’ tinnitus z-scores, a significant positive correlation was obtained between mean bursts per minute and animals’ tinnitus z-scores (r2 = 0.58; P = 0.001; Fig. 4B). However, the mean number of spikes within bursts (r2 = 0.21; P = 0.082; Fig. 4D) and mean burst duration (r2 = 0.10; P = 0.237; Fig. 4F) were not significantly correlated with tinnitus z-scores. This suggests that the changes observed in mean spikes in a burst and mean burst duration during group analysis (i.e. unexposed controls vs. tinnitus animals) may not represent tinnitus-related changes in the burst pattern of MGB units recorded from tinnitus animals.

Increased steepness of rate-level functions of MGB units in tinnitus animals

Single unit discharge rates were measured in response to BBN and CF tones presented randomly across trials at increasing intensities from 0 to 80 dB at 10 dB intervals. Mean single unit BBN rate-level functions were steeper in tinnitus animals (n = 42, six animals) compared to units recorded from unexposed control groups (n = 58, eight animals) (Fig. 5A). Single unit mean firing rates were significantly elevated at higher intensities (unpaired t test, two-tailed; Fig. 5A): 50 dB (P = 0.025), 60 dB (P = 0.002), 70 dB (P = 0.024) and 80 dB (P = 0.049); and at lower intensities: 0 dB (P = 0.001) and 10 dB (P = 0.006). However, MGB unit firing rates between control and exposed animals were not significantly different at 20, 30 and 40 dB.

Figure 5. Rate-level functions comparing average responses of MGB units to broadband noise (BBN) from unexposed control animals (blue; eight animals) with sound-exposed animals with behavioural evidence of tinnitus (red; six animals).

Figure 5

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A–E, rate-level function of MGB units in response to BBN stimuli. A, significant (unpaired t test) increase in average firing rates of MGB units was observed in tinnitus rats (red) at intensities of 0 dB (P = 0.001), 20 dB (P = 0.006), 50 dB (P = 0.025), 60 dB (P = 0.002), 70 dB (P = 0.024) and 80 dB (P = 0.049) compared to control rats (blue). Average firing rates were not significantly different at other intensities. B–E, scatter plots of average firing rates of MGB units (y-axis) from individual animals in response to BBN stimuli at different intensities against that animal's tinnitus z-score. A significant correlation was observed between average firing rates and animals’ tinnitus scores at 60 dB (C) (y = 10.21x + 20.53, P = 0.002), 70 dB (D) (y = 9.707x + 29.13, P = 0.014) and 80 dB (E) (y = 10.39x + 38.92, P = 0.017) but not at 30 dB (B). In all the plots, blue and red squares represent control and sound-exposed animals, respectively. Pearson correlation coefficient was used to determine statistical significance of the correlations. Error bars represent SEM.

Mean BBN-driven rates were directly correlated with tinnitus z-scores at higher stimulus levels: 50 dB (r2 = 0.42; P = 0.010; data not shown), 60 dB (r2 = 0.55; P = 0.002; Fig. 5C), 70 dB (r2 = 0.40; P = 0.014; Fig. 5D), 80 dB (r2 = 0.38; P = 0.017; Fig. 5E); and at lower stimulus levels: 0 dB (r2 = 0.56; P = 0.002; data not shown) and 10 dB (r2 = 0.48; P = 0.006; data not shown). At other intensities, correlations between CF tone discharge rates and tinnitus score were not significant (as represented by 30 dB; r2 = 0.11; P = 0.228; Fig. 5B). Similar to the rate-level function in response to BBN stimuli, the slope of the rate-level function of MGB units in response to their CF tones was also significantly steeper (unpaired t test, two-tailed; Fig. 6A) in tinnitus animals compared to unexposed control animals at 10 dB (P = 0.027), 40 dB (P = 0.048), 50 dB (P = 0.025), 60 dB (P = 0.035), 70 dB (P = 0.036) and 80 dB (P = 0.012), but not at other lower intensities. In addition, positive correlation analysis between mean firing rates in response to CF tone stimuli at individual intensities and animals’ tinnitus z-scores exhibited significant correlations with an animals’ tinnitus score at intensities of 10 dB (r2 = 0.41; P = 0.009; data not shown), 50 dB (r2 = 0.31; P = 0.028; data not shown), 60 dB (r2 = 0.40; P = 0.011; Fig. 6C), 70 dB (r2 = 0.37; P = 0.015; Fig. 6D) and 80 dB (r2 = 0.39; P = 0.012; Fig. 6E) but not at other intensities, represented by 30 dB (r2 = 0.035; P = 0.501; Fig. 6B). These results show that slopes of rate-level functions of MGB units are significantly steeper in tinnitus animals than in unexposed control animals in response to both BBN and unit CF tone stimuli.

Figure 6. Rate-level functions comparing average responses of MGB units to individual unit's CF from unexposed control animals (blue; nine animals) with sound-exposed animals with behavioural evidence of tinnitus (red; six animals).

Figure 6

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A–E, rate-level function of MGB units in response to unit's CF. A, significant increase (unpaired t test) in average firing rates of MGB units was observed at 10 dB (P = 0.027), 40 dB (P = 0.048), 50 dB (P = 0.025), 60 dB (P = 0.035), 70 dB (P = 0.036) and 80 dB (P = 0.012) in tinnitus rats (red) compared to control rats (blue). Average firing rates were not significantly different at intensities of 30 dB and below. B–E, scatter plots of average firing rate of MGB units recorded from individual animals in response to CF stimuli (y-axis) at different intensities against that animal's tinnitus z-score. Significant correlations were observed at 60 dB (C) (y = 10.47x + 20.71, P = 0.011), 70 dB (D) (y = 10.23x + 31.18, P = 0.015) and 80 dB (E) (y = 11.64x + 39.75, P = 0.012) but not at 30 dB (B). In all the plots, blue and red squares represent control and sound-exposed animals, respectively. Pearson correlation coefficient was used to determine statistical significance of the correlations. Error bars represent SEM.

Discussion

The present study compared the single unit discharge properties of MGB neurons, recorded from awake unexposed control rats and sound-exposed rats with behavioural evidence of tinnitus. The tinnitus rats were found to have (1) elevated SFRs (Fig. 3A) and elevated spontaneous bursting activity (Fig. 4A, C and E) of MGB units, and (2) steeper rate-level functions to both BBN (Fig. 5A) and unit CF tones (Fig. 6A). In addition, considering all animals studied, (3) there were significant positive correlations between MGB unit discharge properties (e.g. SFRs, bursting and rate-level slope) and standardized tinnitus scores (Figs 3B, 4B, 5B–E and 6B–E), suggesting that features of MGB unit activity functionally relate to tinnitus and not necessarily the response to sound exposure.

Neural correlates of tinnitus, or high-level sound exposure, such as elevated SFRs, elevated bursting activity, neural synchrony, reorganization of tonotopic maps and steeper rate-level functions/elevated evoked potentials have been previously reported at different levels of the auditory pathway including dorsal cochlear nucleus (DCN) (Brozoski et al. 2002; Kaltenbach et al. 2004; Zhang et al. 2006; Finlayson & Kaltenbach, 2009; Pilati et al. 2012), ventral cochlear nucleus (Vogler et al. 2011), inferior colliculus (IC) (Ma et al. 2006; Bauer et al. 2008; Mulders & Robertson, 2009; Mulders et al. 2010; Longenecker & Galazyuk, 2011; Manzoor et al. 2012,) and auditory cortex (Norena & Eggermont, 2003; Yang et al. 2007; Engineer et al. 2011; Yang et al. 2011), all of which are hypothesized to underpin the tinnitus percept. The present study reports tinnitus-related changes in the discharge properties of MGB neurons from an awake sound-exposed animal model while providing evidence of positive correlations between neural correlates of tinnitus and behavioural tinnitus scores. To our knowledge only Llano et al. (2012) have shown significant correlations between increased cortical activation and gap–startle ratios.

Auditory thalamus as a site for the maintenance of the tinnitus percept

The MGB plays an active role in integrating, shaping and gating the relative salience of auditory representations from upstream (i.e. brainstem-thalamic and midbrain-thalamic information) and downstream auditory and non-auditory information (i.e. cortico-thalamic and limbic-thalamic inputs) (Bartlett & Smith, 1999; Bartlett & Wang, 2007,; Winer et al. 2011; Bartlett, 2013) making it a primary focus for tinnitus pathology (Llinas et al. 1999; Leaver et al. 2011; Zhang, 2013). Several lines of evidence support this contention. Thalamic neurons exhibit two modes of firing, a tonic firing mode that is often seen during brain activated states of waking and rapid eye movement sleep and rhythmic bursting mode observed during slow-wave sleep (Sherman, 2001) and in disorders with positive symptoms such as Parkinson's disease, petit mal epilepsy, tinnitus and chronic pain (Jeanmonod et al. 1996; Llinas et al. 1999; Llinas & Steriade, 2006). In the present study we report similar tinnitus-related elevations in spontaneous bursting activity of MGB units (Fig. 4A and C) and elevated SFRs (Fig. 3A and B), the underlying mechanism of which can be potentially explained using the thalamocortical dysrhythmia hypothesis (Llinas et al. 1999). According to this model, in pathological conditions such as tinnitus, extended hyperpolarization of MGB neurons, due to excess tonic inhibition, de-inactivates T-type Ca2+ channels expressed on MGB neurons, and results in an increase in low threshold bursts. Tonic GABA conductance mediated by extrasynaptic δ-containing GABAA receptors (δ-GABAARs) have been reported to promote burst firing of thalamic relay neurons (Cope et al. 2005; Bright et al. 2007; Brickley & Mody, 2012). Based on the present findings, we hypothesize that changes in the expression level and function of δ-GABAARs may result in increased thalamic bursting. This hypothesis is supported by an in-progress study showing significant tinnitus-related (1) elevation in the extrasynaptic GABAA receptor δ subunit message; (2) maximal evoked current and current density mediated by δ-GABAARs in vitro; and (3) increased number of spikes in a burst recorded from MGB neurons of sound-exposed animals with behavioural of tinnitus (Sametsky et al. 2014). These results suggest that changes in δ-GABAAR levels and function represent one potential mechanism for elevated spontaneous bursting activity and thus increased SFRs of MGB units. Changes in endogenous GABA levels and ionic channelopathies will need to be considered. Additional studies are required to separate ascending excitatory increases impacting neurons in the MGB (Engineer et al. 2011; Kaltenbach, 2011; Richardson et al. 2012) from de novo thalamocortical changes reported here.

In addition to the observed elevations in SFRs and bursting activity, tinnitus rats showed steeper rate-level functions of MGB units in response to BBN and CF tones. Previous studies reported tinnitus-related increases in the output of IC neurons (Ma et al. 2006; Bauer et al. 2008; Mulders & Robertson, 2009; Mulders et al. 2010; Longenecker & Galazyuk, 2011; Manzoor et al. 2012,) that were hypothesized to be, at least in part, the result of an increased input from hyperactive principal output neurons (fusiform cells) of DCN (Manzoor et al. 2013b). The IC is the major upstream auditory input to the MGB and consists of both excitatory (70–80%) and inhibitory (10–30%) projections (Winer et al. 2005; Winer & Lee, 2007). The upstream influence of tinnitus-related hyperactivity in DCN fusiform cells could be potentiated by the tinnitus-related down-regulation of inhibition at the level of the IC (Dong et al. 2010). Increasing tinnitus-related excitatory drive from the IC (Ma et al. 2006; Bauer et al. 2008; Mulders & Robertson, 2009; Mulders et al. 2010; Longenecker & Galazyuk, 2011; Manzoor et al. 2012,) could increase sound-evoked activity of MGB units, possibly underpinning the observed steeper rate-level functions in response to BBN and CF tones in MGB units in tinnitus rats. Furthermore, increased IC excitation onto modestly hyperpolarized MGB neurons could increase activation of T-type Ca2+ channels expressed on MGB neurons, resulting in increased bursting and increased output of the MGB (Llinas et al. 1999; Sametsky et al. 2014).

An alternative explanation for the present findings is the noise-cancellation model proposed by Raushchecker and colleagues (Rauschecker et al. 2010; Leaver et al. 2011). According to this hypothesis, in normal conditions, sound-evoked activity from thalamus is relayed to auditory cortex and further to subcallousal regions through projections from auditory cortex to ventromedial prefrontal cortex and from MGB to nucleus accumbens through the amygdala (Rauschecker et al. 2010; Leaver et al. 2011). The subcallousal region provides feedback excitatory projections onto the thalamic reticular nucleus (TRN) which in turn inhibits MGB neurons acting as a gain control that filters out unpleasant auditory inputs (Rauschecker et al. 2010; Leaver et al. 2011). However, in auditory disorders such as tinnitus, peripheral deafferentation-mediated plastic changes in central auditory structures might lead to an imbalance in excitation–inhibition homeostasis at this MGB–auditory cortex–subcallosal network causing a reduced excitatory drive onto TRN from subcallosal structures and thus reduced inhibitory drive onto MGB neurons leading to altered MGB gating function and elevated discharge properties of MGB units (Muhlau et al. 2006; Rauschecker et al. 2010; Leaver et al. 2011).

In addition to these hypotheses, previous studies have reported sound-induced plastic changes in neuromodulatory systems in various auditory nuclei (Kaltenbach & Zhang, 2007; Engineer et al. 2013; Manzoor et al. 2013a). In this context, a putative role of tinnitus-related plastic changes in neuromodulatory neurotransmitter systems such as cholinergic (Varela & Sherman, 2007; Motts & Schofield, 2011) and serotonergic systems (Varela & Sherman, 2009) that project to MGB and modulate the excitability of MGB neurons cannot be ruled out. Lastly, recent evidence of tinnitus-related changes in the intrinsic properties of DCN neurons (Li et al. 2013) suggests that changes in the biophysical properties of Na+, K+ and Ca2+ conductance that play an important role in regulating the excitability of MGB neurons (Bartlett & Smith, 1999) may also contribute to the observed changes in the discharge properties of MGB neurons.

In conclusion, the present study demonstrates a significant and direct relationship between a behavioural measure of tinnitus in an established sound exposure model and the elevated discharge properties of neurons recorded from the MGB in awake rats. In support of the thalamocortical dysrhythmia hypothesis, MGB units showed tinnitus-dependent increases in SFRs, spontaneous bursting activity and steeper rate-level functions in response to BBN and unit CF tones. Future studies are needed to delineate the underlying mechanisms that underpin elevated discharge properties of MGB units in models of tinnitus.

Acknowledgments

We thank L. Ling, D. Larsen, R. Cai and B. Richardson for their extensive help and support with all aspects of these studies.

Glossary

BBN

broadband noise

CF

characteristic frequency

DCN

dorsal cochlear nucleus

δ-GABAAR

δ-containing GABAA receptor

GPIAS

gap pre-pulse inhibition of acoustic startle

IC

inferior colliculus

MGB

medial geniculate body

PPI

pre-pulse inhibition

SFR

spontaneous firing rate

SPL

sound pressure level

TRN

thalamic reticular nucleus.

Additional information

Competing Interests

None.

Author contributions

B.I.K. performed the experiments; B.I.K., T.J.B., J.T and D.M.C. were involved in designing the experiments, analysis and interpretation of data, preparation of manuscript and approval of the final version of the manuscript. All authors have read and approved the final submission.

Funding

This work was supported by grants from the USA Office of Naval Research # N000141210214 and from the National Institutes of Health (DC000151), both to D.M.C.

Key points

  • Medial geniculate body (MGB) single units recorded from sound-exposed animals with behavioural evidence of tinnitus exhibits enhanced spontaneous firing and burst properties.

  • MGB units in tinnitus animals exhibit increased rate-level function slope when driven by broadband noise and tones at the unit's characteristic frequency.

  • Elevated patterns of neuronal activity and altered bursting showed a significant positive correlation with animals’ tinnitus scores.

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