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Suppression of Auditory Cortical Inhibition Induces Tinnitus

Wesley Jackson Helen Wills Neuroscience Institute University of California, Berkeley

Lab P.I.: Shaowen Bao Cognitive Science Sponsor: David Wessel

Keywords: tinnitus, GABAergic function, inhibition Abstract Tinnitus, the perception of sound without an external stimulus, can have

December 2011

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many causes and associated changes along the auditory pathway. Although

determining which functional changes cause tinnitus has been difficult, the most

likely fundamental mechanism of tinnitus appears to be reduced GABAergic function in the central nervous system from suppression of the GAD65 enzyme. This study seeks to determine whether direct GAD65 suppression in the auditory cortex is

sufficient to cause tinnitus. We found significant auditory cortical GAD65 suppression and tinnitus behavior in mice subjected to either unilateral hearing lesion or viral transfection of Gad2 SiRNA (to directly induce GAD65 suppression), suggesting that tinnitus.

reduced GABAergic function in the auditory cortex may in fact be responsible for Introduction

often caused by damage to the peripheral auditory system, for instance from aging or exposure to loud sound (Eggermont & Roberts, 2004). Such peripheral damage leads to deafferentation of A1 neurons receptive to the impaired auditory input (Kopell & Friedland, 2009, p. 971). It is well understood that sensory deafferentation causes significant changes to the central nervous system. For

Tinnitus is the perception of sound in the absence of external stimuli and is

example, cortical map reorganization in response to deafferentation has been

observed in the visual cortex (Chino et al., 1991) as well as the somatosensory

cortex (Pons et al. 1991). Likewise, the auditory cortex undergoes reorganization following hearing damage (Dietrich et al., 2001). Auditory deafferentation also

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2003) and the dorsal cochlear nucleus (Zhang & Kaltenbach, 1998), as well as

results in increased spontaneous activity in the auditory cortex (Seki & Eggermont, morphological changes to hearing-impaired regions of the cortex and changes in

synaptic plasticity including enhanced excitability and reduced inhibition (Yang et merely emerge from deafferentation has been challenging.

al., 2011). Distinguishing the functional changes that cause tinnitus from those that Although reorganization of the auditory cortex following hearing lesion

(HL) has been implicated as a mechanism of tinnitus and manipulating cortical Okamoto et al. 2010), changes in cortical plasticity do not likely cause tinnitus.

plasticity may have therapeutic benefits in alleviating tinnitus (Engineer et al., 2011;

Hearing lesion-induced cortical reorganization consists of an increase in cortical representation for frequencies below that of the hearing lesion and presumed tinnitus pitch (Yang et al., 2011). However, regions of the auditory cortex previously tuned to frequencies at or above the hearing lesion frequency lose their functional organization and frequency selectivity (Yang et al., 2011). It is unclear why

functional cortical regions with enhanced response to frequencies below that of the that the over-excited and disorganized regions of the cortex generate tinnitus because these regions remain active and may still be interpreted by efferent

hearing lesion and presumed tinnitus percept would be responsible. It is more likely

connections as encoding the frequencies within the hearing-loss or tinnitus percept range. More direct evidence that cortical plasticity is not directly responsible for

complete hearing loss (Su, 2011). This results in complete auditory map deletion as not directly depend on organization of the auditory cortex. Rather, changes in related to tinnitus.

tinnitus is that tinnitus behavior has been observed in an animal model subjected to

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the cortex no longer responds to sound, indicating that the presence of tinnitus may synaptic plasticity due to modulations in tonic inhibition are more likely causally Tinnitus is likely caused by the homeostatic increase in excitability of

deafferented neurons in response to lack of input-driven activity. This may be uptake that controls overall neuron excitability (Richardson, 2009). GABA, a

achieved by reduced tonic inhibition, which is mediated by extrasynaptic GABA primary inhibitory neurotransmitter, is synthesized by glutamic acid decarboxylase

in the hearing-impaired region of the auditory cortex following hearing lesion (Yang et al., 2011). GAD suppression in response to hearing damage has also been observed in the inferior colliculus (Milbrandt et al., 2000). If GAD suppression

(GAD65, hereafter GAD). GAD has been shown to be downregulated, or suppressed,

causes tinnitus, then drugs that enhance inhibition should ameliorate tinnitus.

Indeed, vigabitrin, a GABA agonist, reverses tinnitus behavior in an animal model function should directly mediate tinnitus.

(Brozoski et al., 2006). If tinnitus depends on the efficacy of GABA, then GABAergic This study seeks to determine whether suppressing GAD expression in

and are the primary inhibitory interneurons (Benes & Berretta, 2001). They

GABAergic interneurons can cause tinnitus. GABAergic interneurons produce GAD

constitute about one fifth of the auditory cortex (Potter et al., 2008; Prieto et al.,

1994) and regulate local neuronal activity and synaptic plasticity within the cortex possible by exposing Gad2 siRNA lentivirus to the auditory cortex to prevent (Yuan et al., 2011). Suppressing GAD65 expression in GABAergic interneurons is

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transcription of the GAD gene (GAD-65, 2011). Comparing the behavioral results and GAD expression levels of virally-transfected mice to those of hearing-lesioned mice should indicate whether GAD suppression can be a reliable cause of tinnitus. Materials and Methods Materials 12 Dlx6a-Cre mice (The Jackson Laboratory, Bar Harbor, Maine) aged 30-65

days were trained to an active avoidance task, subjected to either unilateral hearing lesion or viral transfection of Gad2 siRNA lentivirus, and then tested for tinnitus. All experimental procedures were reviewed and approved by the UC Berkeley Animal Care and Use Committee. The test environment was an Acoustic Systems (Austin, Texas) sound attenuation chamber divided by a barrier into two compartments

accessible by a 2x2-inch door opening. A steel rod mesh floor provided shock and a speaker located above and centered between the compartments presented the

13-watt central overhead light equally illuminated both compartments. A 300-watt sound stimuli. Behavioral results were observed and analyzed using LabView 7.1 Anesthesia Protocol

software and National Instruments Data Acquisition (NI-DAQ).

(50mg/kg) and xylazine (10mg/kg) and were placed on a 37C Harvard Apparatus heating pad. Respiratory function and hind-paw withdrawal reflexes were

Mice were anesthetized with intraperitoneal injections of ketamine

monitored to ensure sufficient anesthesia and sedation. Saline and Ringer were periodically administered to maintain hydration during surgery. Auditory Brainstem Response (ABR)

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hearing lesion and 10 days afterwards using BioSigRP software implemented in a Tucker Davis Technology Sys3 recording rig (Alachua, FL). A calibrated earphone and 32 kHz stepping from 70-0 dB at 5dB intervals 19 times per second). Three

Hearing thresholds of anaesthetized mice were measured before unilateral

(TDT) taped to the left ear generated tone trains (3ms full-cycle sine tones of 4, 8, 16 subcutaneous electrodes inserted at the base of each ear and the vertex of the skull recorded the evoked responses. The hearing threshold indicates the lowest sound intensity required to evoke a discernible response. Hearing Lesion (HL)

chamber. Long-term unilateral hearing lesions were induced in the left ear by a Tucker Davis Technology earphone (Alachua, FL). A Bruel and Kjaer 4135 and after hearing lesions. Viral Transfection (V)

Six trained mice were anesthetized and placed in a sound attenuation

continuous 8kHz pure tone at 110dB SPL for two hours generated by a calibrated condenser microphone (Naerum, Denmark) calibrated the sound intensity before

for surgery, during which AI of the right hemisphere was aseptically exposed and

Six trained mice were anesthetized and set in a sound attenuation chamber

injected to cohere with a unilateral hearing lesion of the left ear. 1l of Gad2 siRNA

lentivirus was introduced to cortical layers III and IV 1 via micromanipulator (World Precision Instruments). Active Avoidance Shuttle Behavior Protocol Tinnitus behavior was observed via modified active avoidance shuttle task

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(Jastreboff & Sasaki, 1994; Active, 2011). Mice were habituated for at least two

days prior to training in the test environment in one-hour periods. Then they were trained to actively ambulate across the barrier (shuttle) to avoid a foot-shock of at least 0.4mA presented within seven seconds of the stimulus. Stimuli were one of

seven pure or broadband sounds ranged 4-20kHz at 40, 50, or 60dB to condition the animals to tinnitus-like stimuli 2. The sound ended once the animal made a complete shuttle (the entire body across the barrier, excluding the tail). Training consisted of six 45-55 minute trials per week, for about three weeks. Testing began once for at least three consecutive days. performance (the percent of successful active avoidance shuttles) surpassed 80% Animals were trained for thirty minutes before each testing session. Tests

consisted of nine 60-second probe trials randomly interspersed with 15-25 training observed active avoidance shuttle frequency during silence (NS) and one sound
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trials to maintain satisfactory performance. Eight silent probe (no-sound) trials

About 25% of layer III/IV neurons are GABAergic (Prieto et al., 1994), and these layers receive thalamic and intra-cortical input (ibid; Vaughan & Peters, 1985; Richardson et al., 2009). As deafferentation of thalamic input should directly modulate GABAergic activity in these layers, these layers should be good targets for inducing tinnitus. 2 Although human subjects may vary in their reports of tinnitus perception, tinnitus is most commonly perceived as broadband sound and less commonly as sine tones (Penner, 1995; Tyler et al., 2008).

probe trial observed shuttle frequency during protracted sound stimulus (S). S

that may arise from the stress of surgery or tinnitus (Folmer et al., 2003; Kaltenbach shuttle frequency. This ratio only involved testing sessions of at least 75% active 2010; Sullivan et al., 1993). NS/S indicates how strongly constant sound potentiates

conditions; it also insures against potential confounds from depressive behavior

serves two functions: it is a model of expected shuttle frequency in tinnitus-like

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avoidance performance to prevent confounds from poor performance. This initial followed by unilateral hearing lesion or exposure of Gad2 siRNA lentivirus to the auditory cortex.

testing phase ended once an individuals NS/S was consistent for at least four days,

training to ensure performance of at least 80%. Testing then resumed to measure changes in shuttle frequency. Because tinnitus should cause silent-probe trial shuttle frequency to approach that of sound-probe trials, NS/S should be

Mice recovered for at least 11 days after viral transfection before resuming

significantly increased if the animal experiences tinnitus. It is expected that these positively correlate with the percent increase in tinnitus-motivated behavior values).

results agree with GAD expression such that the percent of GAD suppression should (indicated by NS after tinnitus-induction compared to nave, or pre-induction, Quantification of Gene Expression GAD expression was quantified using RT-PCR. RNA samples from nave and

lesioned regions of the auditory cortex were prepared with TRIzol reagent (Ambion, transcribed 3g of RNA, with 18S rRNA as an internal standard. 50l of PCR mixture

Austin, TX). A first-strand cDNA synthesis kit (BD Biosciences, Palo Alto, CA) reverse

contained 10X Taq buffer, 0.3 U Taq DNA Polymerase (QIAGEN, Valencia, CA), 2.5M of dNTPs, 5pmol of primers, and 50mg of cDNA template from each auditory cortex. PCR reactions underwent initial denaturation at 94C for five minutes followed by 25-35 cycles at 94C for 30 seconds, 62-65C depending on the primers for 30 seconds, and 72C for 60 seconds. Amplification was optimized to prevent

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saturation of amplified bands. PCR products were quantified via electrophoresis in 1.5% agarose gel and stained with ethidium bromide. Band intensities were measured with BIORAD Gel Doc 2000 (Bio-Rad Laboratories, Hercules, CA). Results Hearing Lesion Increases Hearing Thresholds Figure 1A shows that unilateral hearing lesion silenced ABR for the lesioned

32kHz, but there was no change for the preserved ear (mean threshold: lesioned ear pre-HL: 35.9dB 7.4, lesioned ear post-HL: 67.1dB 1.7, p < 0.01; preserved ear pre-HL: 37dB 7.4, preserved ear post-HL: 35.4dB 7.6, p = 0.75). Behavior Hearing lesion and viral transfection significantly, though differentially, Hearing Lesion and Viral Transfection Result in GAD Suppression and Tinnitus

ear and 1B demonstrates a significant increase in hearing thresholds across 4-

suppressed GAD expression in the auditory cortex (HL: mean suppression: 52.78% 0.72, p < 0.01; V: mean suppression: 62.75% 13.0, p < 0.05; HL versus V: p = 0.26). Figure 2 shows post-hearing lesion GAD quantification results. Because both hearing lesion and viral transfection significantly suppress GAD expression, the behavioral results should indicate tinnitus behavior in both cases.

behavior and Figure 4 indicates the effects of hearing lesion and viral transfection on shuttle behavior. Hearing lesion had no effect on active shuttle performance (Figure 4Ai; nave: 84.8% 1.7, HL: 83.9% 2.8, p = 0.8), but caused tinnitus-

Figure 3 illustrates the paradigm for associating tinnitus with active shuttle

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indicating behavior. Post-hearing lesion shuttle frequencies were normalized with

respect to nave frequencies (valued at 100) to determine general changes in shuttle frequency despite individual variation in overall activity. NS increased (Figure 4Bi; 9.2, p < 0.01). Thus the post-HL ratio NS/S significantly increased (Figure 4Ci; Like the hearing lesion, viral transfection did not influence performance normalized mean: 141% 15.2, p < 0.01) while S decreased (normalized mean: 64% mean nave: 0.27 0.04, mean HL: 0.61 0.07, p < 0.01).

(Figure 4Aii; nave: 82.9% 3.1, V: 83% 2.8, p = 1.), yet resulted in tinnitus

< 0.01) and was positively correlated with GAD suppression (Figure 5 shows a logistic curve fit to this data; R2 = 0.72, RMSE = 22.7). This amplification of NS

behavior. NS significantly increased (Figure 4Bii; normalized mean: 173.6% 11, p

appears to approach a limit of about 200% after GAD expression has been reduced 0.16). Although hearing lesion attenuated S, viral transfection had no effect on sound-shuttle frequency (normalized mean: 95.9% 17, p = 0.82). Still, viral

by 40%. Viral transfection more significantly potentiated NS than hearing lesion (p =

transfection significantly increased NS/S (Figure 4Cii; mean nave: 0.3 0.05, mean correlated with that from hearing lesion (p = 0.82). Statistics V: 0.58 0.12, p < 0.05). The potentiation of NS/S from viral transfection is not

behavioral results and the ANOVA (Tukey unpaired t) test was used for analyzing were normalized to 100 to determine changes following the experimental Discussion

The paired t-test was used to determine the statistical significance of

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ABR. Nave active shuttle frequencies (pre-hearing lesion and pre-viral transfection)

conditions. 5% significance levels were used. Data is presented as mean SEM. It is well established that active avoidance behavior can be a reliable

indicator of tinnitus regardless of how the tinnitus is induced (e.g. by drugs or

hearing lesion) (Guitton et al., 2003; Jastreboff & Sasaki, 1994; Yang et al., 2011).

Arguably, tinnitus can have such varied causes because drugs and hearing lesions activity as a potential fundamental cause of tinnitus and demonstrates that viral in an animal model comparable to that induced by hearing lesion.

reduce inhibitory activity in the auditory system. This study targets such inhibitory vectors can disrupt auditory cortical GABAergic function to induce tinnitus behavior As intended, the unilateral hearing lesion induced severe unilateral hearing

damage, significantly elevating hearing thresholds obtained from the lesioned ear across 4-32kHz. The influence of viral transfection on hearing thresholds was not the hearing lesion rather than determining the successful induction of tinnitus. However, viral transfection should have had no effect on hearing thresholds 3.
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tested for in this study because ABR results are useful for determining the success of

Salicylate causes tinnitus and increases hearing thresholds without modulating

GAD suppression is a purported effect of hearing damage rather than a cause of it.

cortical GAD expression, but it acts on peripheral and midbrain structures (Bancroft

Indeed, comparison of active shuttle frequencies in hearing lesioned versus virally transfected mice suggests significant differences in the effects of each induction method beyond tinnitus. As expected, both hearing lesion and viral transfection increased the no-

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sound shuttle frequency, indicating tinnitus. This may be simply attributed to the animals failure to distinguish between their subjective tinnitus and the external sound stimulus in both cases. The apparent limit on this tinnitus-driven crossing maximal shuttle frequency due to fatigue as opposed to a limit on the efficacy of

behavior for virally-transfected mice (as indicated in Figure 5) likely represents a

virally-mediated GAD suppression specifically. This is supported by the fact that

than hearing lesion, as well as a greater suppression of GAD, although both methods appear sufficient to induce tinnitus in this animal model. It is important to explain the differences in sound-shuttle frequency between

viral transfection had a significantly greater effect on silent-probe shuttle frequency

hearing lesion and viral transfection, as this appears to be the main factor

differentiating their NS/S, or tinnitus-indicating, results. Unlike the hearing lesion, viral transfection did not significantly influence sound shuttle frequency. It is difficult to determine why hearing lesion in particular would decrease shuttle

frequency during constant sound, but ostensibly could be due to painful hyperacusis et al., 1991; Guitton et al. 2003). It likely simulates a hearing lesion and causes tinnitus via activation of NMDA receptors in the outer hair cells of the cochlea (Guitton 2003; Puel 2007).

inflicted by the lesion. Hyperacusis is an increased sensitivity to sound and is often indicating the influence of hyperacusis on active avoidance behavior, but because stress can cause depressive behavior in the form of reduced anhedonia (painaversion) (Duric et al., 2010), pain from the constant-sound stimulus may reduce these tinnitus-induction methods do not pose an apparent challenge for this painful, leading to sound aversion (hyperacusis, 2006). There is yet no research

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active avoidance motivation. Although differences in the behavioral consequences of behavioral paradigm (as tinnitus was indicated in both cases), determining reasons for such differences in sound-probe shuttle behavior may be an interesting avenue for future research.

animal model appears to be a satisfactory general model of tinnitus regardless of

Because NS/S is amplified following hearing lesion or viral transfection, this

how the tinnitus is induced. This is corroborated by the fact that despite differences methods, both methods resulted in tinnitus-behavior with significant GAD

in shuttle frequency and GAD suppression efficacy between these tinnitus-induction suppression, as expected. It is of note, however, that there appear to be clear distinctions in the reliability of each method as a means of inducing tinnitus. GAD suppression, it was difficult to ensure consistent suppression via viral Although unilateral hearing lesion appears to produce reliable and consistent

transfection, as indicated by its significantly larger variance in suppression percent. This is likely because of the difficulty in consistently locating the target of the virus during surgery for each mouse. Thus, viral transfection may not be a good

therapeutic option for tinnitus sufferers unless more consistent manipulations of auditory cortical GABAergic function are developed. Despite the differential behavioral consequences and GAD suppression by

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hearing lesion and viral transfection of Gad2 SiRNA, significant GAD suppression consistently correlated with potentiated tinnitus behavior, indicating that GAD can cause tinnitus, it is expected that increasing GAD expression (e.g. via viral

suppression is in fact a sufficient cause of tinnitus. Because direct GAD suppression vectors) should alleviate tinnitus, regardless of how the tinnitus was acquired. This would help establish that GABAergic function is the fundamental mechanism of tinnitus. References

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Figure 1. Unilateral hearing lesion increases the hearing threshold. A. Tone pips (3ms full-cycle sine waves of 4, 8, 16 and 32kHz ranging from 70-0dB in 5dB decrements) were presented binaurally to elicit auditory brainstem responses

(ABR). Hearing lesion silences ABR of the hearing-lesioned (left) ear. B. Hearing

threshold was determined to be the smallest dB value required to elicit a

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for the lesioned ear. There is no significant change in ABR or hearing threshold for the hearing-preserved (right) ear.

discernable response. Hearing lesion increases hearing threshold across 4-32kHz

Figure 2. Hearing lesion reduces GAD expression. A. Photomicrographs reveal decreased GAD levels in the contralateral auditory cortex following unilateral 18SrRNA values. hearing lesion. B. GAD expression was normalized with respect to corresponding

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shuttle across a barrier to avoid shock when presented with a sound stimulus

Figure 3. Active-avoidance shuttle behavior paradigm. Mice were trained to actively

performance reached 80% for three consecutive days. Test trials consisted of 60 the barrier and respective shuttle frequencies were recorded. The ratio of no-

presented every 40-70s. Test probe trials were introduced after active avoidance

seconds of sound or silence, during which mice were allowed to freely shuttle across sound/sound shuttle frequency indicates the influence of persistent sound on transfection of Gad2 siRNA suggests that tinnitus motivates no-sound shuttle behavior.

shuttle behavior. A significant increase in this ratio after hearing lesion or viral

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Figure 4. Hearing lesion and viral transfection of Gad2 siRNA result in tinnitus

behavior. Ai,ii. In either case, there was no significance change in active avoidance performance. Bi. Unilateral hearing lesion potentiates no-sound shuttle frequency NS but does not alter S. Ci,ii. Hearing lesion and viral transfection significantly increase NS/S ratio. (NS) and suppresses sound shuttle frequency (S). Bii. Viral transfection potentiates

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Figure 5. GAD suppression potentiates no-sound shuttle frequency, which determined by normalizing GAD expression levels against their respective

approaches a limit near 200%. Knockdown, or suppression, percentage was unaffected values in the contralateral auditory cortex. A logistic curve fits the data well (R2 = 0.72, RMSE = 22.7, indicating a limited positive correlation between tinnitus-driven shuttle behavior and GAD suppression.