PROCEEDINGS of the23rd International Congress on Acoustics
9 to 13 September 2019 in Aachen, Germany

Targeted neuroplasticity in rat primary auditory cortex with vagus
nerve stimulation and near-threshold tones
Alan M. CARROLL1; Michael P. KILGARD1
1

The University of Texas at Dallas, United States

ABSTRACT
Vagus nerve stimulation (VNS) is a method for driving therapeutic, targeted neuroplasticity in clinicalpopulations suffering from tinnitus and stroke. VNS facilitates specific cortical changes through the phasic
release of plasticity promoting neuromodulators simultaneously paired with delivery of a sensory stimulus,
such as tones or speech. Recent clinical evidence and ongoing pre-clinical experiments in rats show that VNS
paired with near-threshold somatosensation of the hand/paw can significantly reduce elevated sensory
thresholds resulting from neural injuries after only one week of therapy. A possible explanation for this quick
and robust recovery is that VNS is more effective at driving neuroplasticity in cortical circuits when pairedwith stimuli just above the response threshold of neural receptive fields. To date, all auditory VNS therapies
have used stimuli considerably above auditory thresholds, potentially diminishing the therapeutic effect of
VNS-paired treatments. To test the effectiveness of VNS-paired, near-threshold stimuli in driving auditory
neuroplasticity, unimpaired adult rats will receive VNS repeatedly paired with the brief presentation of a 10dB SPL 9 kHz tone for one week. A cortical map of receptive field properties in primary auditory cortex will
be made one day later and compared to the maps of naïve rats.
Keywords: Vagus nerve stimulation, Auditory cortex, Neuroplasticity

1. INTRODUCTION
Vagus nerve stimulation (VNS) drives phasic release of neuromodulators l ike norepinephrine (1),and can lead to stimulus-specific, long-term changes in auditory receptive fields when repeatedly
paired with the presentation of an auditory stimulus (2–7). Pairing VNS with the presentation of a brief
9 kHz tone increases the cortical representation of 9 kHz in primary auditory cortex (A1), while
pairing with a 19 kHz tone increases the representation of 19 kHz (2). Stimulus-specific plasticity has
also been observed for VNS-paired speech, tone trains, and motor skill learning (3,8,9).
This ability to drive targeted neuroplasticity may enable the treatment of clinical disorders
associated with maladaptive changes in neural circuitry (10,11). A VNS based tonal therapy in a rat
model of tinnitus has been shown to reverse abnormal auditory processing in A1 and re-normalize
behavioral correlates of tinnitus perception. The therapy consisted of repeatedly pairing VNS with thepresentation of a variety of tones with frequencies surrounding (and excluding) the putative tinnitus
frequency (2). A clinical trial however only found a modest improvement of VNS tonal therapy on theTinnitus Handicap Inventory (THI) for a subgroup of chronic tinnitus patients (12). While these initial
results are promising, it is expected that significant improvements in treatment can be made throughthe refinement of therapy design. An obvious target for further investigation is the set of tonal stimuli
used during therapy, as it is unlikely that they optimally drive the necessary neuroplastici ty fortherapeutic benefit.
A recent case study, as well as new preclinical research, provide novel evidence that a simple
change to the tonal stimulus set may enhance targeted plasticity treatment and lead to improved
clinical results. During the follow-up phase of a VNS clinical trial for motor rehabilitation after stroke,a patient underwent additional tactile therapy in an attempt to ameliorate remaining sensory
impairment in his hand. After only ten days of therapy, the patient had an 80% recovery in t actile
threshold (13). Ongoing preclinical experiments in a rat model of sensory deficit have replicated therecovery of sensory threshold in only one week of VNS pairing (unpublished). In both cases, the tactile
therapies consisted of stimuli at the threshold of sensory detection -- in effect driving activity from1alan.carroll@utdallas.edu

6607

only a small subset of the neural population in primary sensory cortex. This is a key differencecompared to the VNS-paired tones used in the tinnitus therapies, which were instead presented atintensities well-above auditory threshold (2,12). It is possible that pairing VNS with tones nearauditory thresholds will be more effective in driving plasticity than more intense tones that alreadydrive significant neural activity.
This study examines the effectiveness of one week of VNS paired with a near-threshold tone (9 kHz
10 dB SPL) in driving stimulus-specific plasticity in rat A1. Previous VNS tone-pairing studies have
all utilized four weeks of treatment with tones well-above the rat auditory threshold (2,4–7). Therefore,
it is unknown if pairing VNS with a near-threshold tone for only a single week will be sufficient toresult in significant neuroplasticity of A1 in a healthy subject (as suggested by the current
somatosensory results in impaired models). This is a crucial first experiment to determine the validityof potential improvements to the stimulus set of future clinical VNS -based therapies.

2. METHODS
2.1 Subjects
Seventeen age-matched, female Sprague-Dawley rats were randomly assigned to either naive(n=11) or VNS (n=6) groups.
2.2 Tone stimulus
VNS was paired with the brief presentation of a 500 ms, 9 kHz, 10 dB SPL tone. Previous studies
suggest that a 9 kHz 10 dB tone recruits about 1/4 the amount of A1 (~10 -15%) as a 9 kHz 50-60 dB
tone (2,4–7).
2.3 Vagus nerve stimulation
VNS subjects had a custom, platinum-iridium bipolar cuff electrode implanted around the left
cervical vagus nerve as described previously (2,4–7). Parameters used for stimulation were the same asprevious studies (2). Briefly, a single stimulation event consisted of a 500 ms train of biphasic pulses(100 us pulsewidth) at a rate of 30 Hz and an intensity of 0.8 mA.
2.4 Pairing procedure
After recovering for seven days from cuff implantation, VNS subjects received five sess ions ofVNS tone-pairing (one session per day). Each session, the subject was placed into a wire cage inside ofa double-walled, sound attenuated booth, 25 cm from a speaker calibrated to play a 9 kHz 10 dB SPL
tone, and plugged into a stimulator by a free-hanging swivel. The 9 kHz tone was presented
approximately every 30 seconds, paired temporally with a VNS event. Each session consisted of 300
pairings and lasted approximately 2.5 hours. At the end of a session, the subject was returned to herhome cage.
2.5 Cortical mapping
Twenty-four hours after the last pairing session, a cortical map of the subject's A1 was made, asdescribed previously (2,4–7). Briefly, the subject was anesthetized with sodium pentobarbital (50mg/kg), and a craniotomy and durotomy was performed to expose right A1. Four parylene -coatedtungsten microelectrodes were inserted into cortex approximately 600 -700 um below the pial surface,and multi-unit neural activity (MUA) was recorded in response to the presentation of tones spanning
1-32 kHz and 0-75 dB SPL from a free-field speaker 10 cm from the left ear. Once recording finished,
the electrodes were inserted into another cortical location. This procedure was repeated systematically
until A1 was completely bordered by non-responsive and/or non-A1 auditory sites.
2.6 Analysis
Each electrode site in a subject’s map was classified as belonging to A1 or not and was considered
representative of the surrounding neural area based on a Voronoi diagram of the points, as performed
in previous studies (14). The average amount of A1 responding to each frequency/intensity tone pairpresented during cortical mapping was then calculated based on the area represented by each electrodesite and represented as a percentage.
The auditory threshold for each site was defined as the lowest intensity that evoked a reliable neuralresponse.

6608

3. Results
One week of VNS pairing with the near-threshold 9 kHz tone successfully drove stimulus-specificplasticity in A1 as compared to a naïve control group. The difference plot in Figure 1 shows a generalincrease in the amount of A1 representation for mid-frequency (4-16 kHz) tones, peaked around 9 kHz.
There is also a strong increase in the representation of low-intensity (0-15 dB) tones across the 1-32
kHz frequency spectrum. These results are comparable to previous VNS tone -pairing experiments
demonstrating stimulus-specific plasticity over four weeks of treatment (4–7). Figure 2 highlights thespecific increase in A1 representation for 10 dB tones between 8-16 kHz for the VNS group comparedto naives (unpaired t-test, p<0.005). The percentage area of A1 responding to 50 dB 8-16 kHz tones isalso plotted for comparison. Although there is a trend for a representational increase in the VNS groupcompared to naives at 50dB also, it is not significant and is within the expected range of A1
representation based on data from previous studies (unpaired t-test, p=0.06) (4–7). Figure 3 shows thegeneral decrease in auditory thresholds across A1 for the VNS group compared to naives (unpairedt-test, p<0.05).

Figure 1 – Difference plot of the percentage of A1 responding between VNS and Naïve groups. The VNSgroup on average had more A1 sites respond with neural activity to the presentation of tones at nearly all
intensities in the frequency range of 4-16 kHz. Additionally, the VNS group has a general increase in theamount of A1 responding at low intensities across all frequencies compared to Naïve. This is strong evidence
that pairing a near-threshold auditory stimulus with VNS for one week is sufficient for driving
stimulus-specific plasticity comparable to what is seen in previous studies.

6609

Figure 2 – Mean percentage of A1 responding specifically to 10 or 50 dB tones within the 8-16 kHz
frequency range. The VNS group has more representation of 8-16 kHz at 10 dB than the Naïve group does(p<0.005). There is a noticeable trend for the VNS group to have more A1 representation at 50 dB also, but it
does not quite exceed what might be expected from a typical Naïve subject (p=0.06)

Figure 3 – VNS pairing with a near-threshold stimulus reduces overall auditory thresholds in A1 (p<0.05)6610

4. CONCLUSIONSVagus nerve stimulation (VNS) is a potential clinical therapeutic tool for treating a wide range ofdisorders associated with maladaptive plasticity, such as tinnitus, through its ability to drive targeted,stimulus-specific neuroplasticity. Although there has been modest success treating tinnitus in initialclinical trials, both the number of patients successfully treated, and the amount of benefit gained fromtreatment may be improved with changes to the therapy stimulus set. Recent evidence suggests thatpairing VNS with stimuli near the threshold of neural receptive fields can drive significant
neuroplasticity in only one week and may be a better therapeutic approach than using stimuli wellabove thresholds. The current study demonstrates that one week of pairing VNS with a near-threshold
9 kHz 10 dB tone drives stimulus-specific plasticity comparable to previous studies using much more
intense auditory stimuli over four weeks of treatment. Future work will focus on comparing theseresults with one week pairing of other stimuli to see if the results are specific to the use of anear-threshold tone.

ACKNOWLEDGEMENTS
This work was sponsored by the Defense Advanced Research Projects Agency (DARPA)
Biological Technologies Office (BTO) TNT program under the auspices of Dr. Doug Weber andTristan McClure-Begley through the Space and Naval Warfare Systems Center, Pacific Grant/Contract
No. N66001-17-2-4011.
Any opinions, findings, and conclusions or recommendations expressed in this publication arethose of the author(s) and do not necessarily reflect the views of the Defense Advanced Research
Projects Agency (DARPA) Biological Technologies Office (BTO).

REFERENCES
1. Hulsey DR, Riley JR, Loerwald KW, Rennaker RL, Kilgard MP, Hays SA. Parametric characterizationof neural activity in the locus coeruleus in response to vagus nerve stimulation. Experimental Neurology.
289:21–30.
2. Engineer ND, Riley JR, Seale JD, Vrana WA, Shetake JA, Sudanagunta SP, et al. Reversing
pathological neural activity using targeted plasticity. Nature. 2011;470(7332):nature09656.
3. Engineer CT, Engineer ND, Riley JR, Seale JD, Kilgard MP. Pairing Speech Sounds With Vagus Nerve
Stimulation Drives Stimulus-specific Cortical Plasticity. Brain Stimul. 2015;8(3):637–44.
4. Borland, Vrana WA, Moreno NA, Fogarty EA, Buell EP, Sharma P, et al. Cortical Map Plasticity as aFunction of Vagus Nerve Stimulation Intensity. Brain Stimulation. 9(1):117–23.
5. Borland MS, Engineer CT, Vrana WA, Moreno NA, Engineer ND, Vanneste S, et al. The Interval
Between VNS-Tone Pairings Determines the Extent of Cortical Map Plasticity. Neuroscience.
2018;369:76–86.
6. Buell EP, Loerwald KW, Engineer CT, Borland, Buell JM, Kelly CA, et al. Cortical Map Plasticity as aFunction of Vagus Nerve Stimulation Rate. Brain Stimulation. 2018;
7. Loerwald KW, Borland MS, Rennaker RL, Hays SA, Kilgard MP. The interaction of pulse width andcurrent intensity on the extent of cortical plasticity evoked by vagus nerve stimulation. Brain Stimulation.
2017;
8. Engineer CT, Shetake JA, Engineer ND, Vrana WA, Wolf JT, Kilgard MP. Temporal plasticity inauditory cortex improves neural discrimination of speech sounds. Brain Stimul. 2017;10(3):543–52.
9. Hulsey DR, Hays SA, Khodaparast N, Ruiz A, Das P, Rennaker RL, et al. Reorganization of Motor6611

Cortex by Vagus Nerve Stimulation Requires Cholinergic Innervation. Brain Stimulation. 9(2).
10. Engineer CT, Hays SA, Kilgard MP. Vagus nerve stimulation as a potential adjuvant to behavioral
therapy for autism and other neurodevelopmental disorders. Journal of Neurodevelopmental Disorders.
2017;9(1):20.
11. Hays SA, Rennaker RL, Kilgard MP. Targeting plasticity with vagus nerve stimulation to treat
neurological disease. Progress in Brain Research. 207.
12. Tyler R, Cacace A, Stocking C, Tarver B, Engineer N, Martin J, et al. Vagus Nerve Stimulation Pairedwith Tones for the Treatment of Tinnitus: A Prospective Randomized Double-blind Controlled Pilot
Study in Humans. Sci Rep-uk. 2017;7(1):11960.
13. Kilgard MP, Rennaker RL, Alexander J, Dawson J. Vagus nerve stimulation paired with tactile
training improved sensory function in a chronic stroke patient. Neurorehabilitation. 2018;42(2):159–65.
14. Kilgard M. Cortical Map Reorganization Enabled by Nucleus Basalis Activity. Science.
279(5357):1714–8.

6612