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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

December 2011

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Keywords: tinnitus, GABAergic function, inhibition

Abstract

Tinnitus, the perception of sound without an external stimulus, can have

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

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

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

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

example, cortical map reorganization in response to deafferentation has been

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

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

results in increased spontaneous activity in the auditory cortex (Seki & Eggermont,

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

morphological changes to hearing-impaired regions of the cortex and changes in

synaptic plasticity including enhanced excitability and reduced inhibition (Yang et

al., 2011). Distinguishing the functional changes that cause tinnitus from those that

merely emerge from deafferentation has been challenging.

Although reorganization of the auditory cortex following hearing lesion

(HL) has been implicated as a mechanism of tinnitus and manipulating cortical

plasticity may have therapeutic benefits in alleviating tinnitus (Engineer et al., 2011;
Okamoto et al. 2010), changes in cortical plasticity do not likely cause tinnitus.

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

hearing lesion and presumed tinnitus percept would be responsible. It is more likely
that the over-excited and disorganized regions of the cortex generate tinnitus
because these regions remain active and may still be interpreted by efferent

connections as encoding the frequencies within the hearing-loss or tinnitus percept

range. More direct evidence that cortical plasticity is not directly responsible for

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

complete hearing loss (Su, 2011). This results in complete auditory map deletion as

the cortex no longer responds to sound, indicating that the presence of tinnitus may
not directly depend on organization of the auditory cortex. Rather, changes in

synaptic plasticity due to modulations in tonic inhibition are more likely causally
related to tinnitus.

Tinnitus is likely caused by the homeostatic increase in excitability of

deafferented neurons in response to lack of input-driven activity. This may be

achieved by reduced tonic inhibition, which is mediated by extrasynaptic GABA

uptake that controls overall neuron excitability (Richardson, 2009). GABA, a

primary inhibitory neurotransmitter, is synthesized by glutamic acid decarboxylase

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

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

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

Indeed, vigabitrin, a GABA agonist, reverses tinnitus behavior in an animal model

(Brozoski et al., 2006). If tinnitus depends on the efficacy of GABA, then GABAergic
function should directly mediate tinnitus.

This study seeks to determine whether suppressing GAD expression in

GABAergic interneurons can cause tinnitus. GABAergic interneurons produce GAD

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

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

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1994) and regulate local neuronal activity and synaptic plasticity within the cortex
(Yuan et al., 2011). Suppressing GAD65 expression in GABAergic interneurons is

possible by exposing Gad2 siRNA lentivirus to the auditory cortex to prevent

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

13-watt central overhead light equally illuminated both compartments. A 300-watt

speaker located above and centered between the compartments presented the

sound stimuli. Behavioral results were observed and analyzed using LabView 7.1

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

Anesthesia Protocol

Mice were anesthetized with intraperitoneal injections of ketamine

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

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

Hearing thresholds of anaesthetized mice were measured before unilateral

hearing lesion and 10 days afterwards using BioSigRP software implemented in a

Tucker Davis Technology Sys3 recording rig (Alachua, FL). A calibrated earphone

(TDT) taped to the left ear generated tone trains (3ms full-cycle sine tones of 4, 8, 16
and 32 kHz stepping from 70-0 dB at 5dB intervals 19 times per second). Three

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)

Six trained mice were anesthetized and placed in a sound attenuation

chamber. Long-term unilateral hearing lesions were induced in the left ear by a

continuous 8kHz pure tone at 110dB SPL for two hours generated by a calibrated
Tucker Davis Technology earphone (Alachua, FL). A Bruel and Kjaer 4135

condenser microphone (Naerum, Denmark) calibrated the sound intensity before

and after hearing lesions.
Viral Transfection (V)

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

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

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

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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

(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

performance (the percent of successful active avoidance shuttles) surpassed 80%

for at least three consecutive days.

Animals were trained for thirty minutes before each testing session. Tests

consisted of nine 60-second probe trials randomly interspersed with 15-25 training
trials to maintain satisfactory performance. Eight silent probe (no-sound) trials

observed active avoidance shuttle frequency during silence (NS) and one sound
probe trial observed shuttle frequency during protracted sound stimulus (S). S

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).
1

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

conditions; it also insures against potential confounds from depressive behavior

that may arise from the stress of surgery or tinnitus (Folmer et al., 2003; Kaltenbach
2010; Sullivan et al., 1993). NS/S indicates how strongly constant sound potentiates

shuttle frequency. This ratio only involved testing sessions of at least 75% active

avoidance performance to prevent confounds from poor performance. This initial

testing phase ended once an individuals NS/S was consistent for at least four days,
followed by unilateral hearing lesion or exposure of Gad2 siRNA lentivirus to the
auditory cortex.

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

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

significantly increased if the animal experiences tinnitus. It is expected that these

results agree with GAD expression such that the percent of GAD suppression should
positively correlate with the percent increase in tinnitus-motivated behavior

(indicated by NS after tinnitus-induction compared to nave, or pre-induction,

values).

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,

Austin, TX). A first-strand cDNA synthesis kit (BD Biosciences, Palo Alto, CA) reverse
transcribed 3g of RNA, with 18S rRNA as an internal standard. 50l of PCR mixture

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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

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

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

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).

Hearing Lesion and Viral Transfection Result in GAD Suppression and Tinnitus
Behavior
Hearing lesion and viral transfection significantly, though differentially,

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.

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Figure 3 illustrates the paradigm for associating tinnitus with active shuttle

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-

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;

normalized mean: 141% 15.2, p < 0.01) while S decreased (normalized mean: 64%
9.2, p < 0.01). Thus the post-HL ratio NS/S significantly increased (Figure 4Ci;

mean nave: 0.27 0.04, mean HL: 0.61 0.07, p < 0.01).

Like the hearing lesion, viral transfection did not influence performance

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

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

< 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

appears to approach a limit of about 200% after GAD expression has been reduced

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

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

transfection significantly increased NS/S (Figure 4Cii; mean nave: 0.3 0.05, mean
V: 0.58 0.12, p < 0.05). The potentiation of NS/S from viral transfection is not

correlated with that from hearing lesion (p = 0.82).

Statistics

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

behavioral results and the ANOVA (Tukey unpaired t) test was used for analyzing

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

were normalized to 100 to determine changes following the experimental

conditions. 5% significance levels were used. Data is presented as mean SEM.

Discussion

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

reduce inhibitory activity in the auditory system. This study targets such inhibitory
activity as a potential fundamental cause of tinnitus and demonstrates that viral

vectors can disrupt auditory cortical GABAergic function to induce tinnitus behavior

in an animal model comparable to that induced by hearing lesion.

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

tested for in this study because ABR results are useful for determining the success of
the hearing lesion rather than determining the successful induction of tinnitus.
However, viral transfection should have had no effect on hearing thresholds 3.
3

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

Salicylate causes tinnitus and increases hearing thresholds without modulating

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

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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-

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

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

maximal shuttle frequency due to fatigue as opposed to a limit on the efficacy of

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

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

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

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).

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inflicted by the lesion. Hyperacusis is an increased sensitivity to sound and is often

painful, leading to sound aversion (hyperacusis, 2006). There is yet no research

indicating the influence of hyperacusis on active avoidance behavior, but because

stress can cause depressive behavior in the form of reduced anhedonia (pain-

aversion) (Duric et al., 2010), pain from the constant-sound stimulus may reduce

active avoidance motivation. Although differences in the behavioral consequences of

these tinnitus-induction methods do not pose an apparent challenge for this

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.

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

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

how the tinnitus is induced. This is corroborated by the fact that despite differences

in shuttle frequency and GAD suppression efficacy between these tinnitus-induction

methods, both methods resulted in tinnitus-behavior with significant GAD

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.

Although unilateral hearing lesion appears to produce reliable and consistent

GAD suppression, it was difficult to ensure consistent suppression via viral

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

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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

hearing lesion and viral transfection of Gad2 SiRNA, significant GAD suppression
consistently correlated with potentiated tinnitus behavior, indicating that GAD

suppression is in fact a sufficient cause of tinnitus. Because direct GAD suppression

can cause tinnitus, it is expected that increasing GAD expression (e.g. via viral

vectors) should alleviate tinnitus, regardless of how the tinnitus was acquired. This
would help establish that GABAergic function is the fundamental mechanism of
tinnitus.

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

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

Figure 2. Hearing lesion reduces GAD expression. A. Photomicrographs reveal

decreased GAD levels in the contralateral auditory cortex following unilateral

hearing lesion. B. GAD expression was normalized with respect to corresponding

18SrRNA values.

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

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

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

performance reached 80% for three consecutive days. Test trials consisted of 60

seconds of sound or silence, during which mice were allowed to freely shuttle across
the barrier and respective shuttle frequencies were recorded. The ratio of no-

sound/sound shuttle frequency indicates the influence of persistent sound on

shuttle behavior. A significant increase in this ratio after hearing lesion or viral
transfection of Gad2 siRNA suggests that tinnitus motivates no-sound shuttle
behavior.

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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) and suppresses sound shuttle frequency (S). Bii. Viral transfection potentiates
NS but does not alter S. Ci,ii. Hearing lesion and viral transfection significantly
increase NS/S ratio.

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Figure 5. GAD suppression potentiates no-sound shuttle frequency, which

approaches a limit near 200%. Knockdown, or suppression, percentage was

determined by normalizing GAD expression levels against their respective

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.