Treatment of Tinnitus with Cholecystokinin-mediated Neuroplasticity

Abstract

Tinnitus is a disease of the auditory system when phantom sounds like hissing, clicking or buzzing are perceived by an individual. In more severe cases these sounds are present constantly throughout the day and are perceived as considerably loud so that they deteriorate the quality of the subject's life. In the worst cases, these phantom perceptions make the patients incapable to perform even basic day-to-day activities. Despite enormous research effort, no officially approved treatment is presently available to treat tinnitus patients.

Neurons along the central auditory pathway show tonotopic organization. They tend to be 'tuned' to a certain frequency called characteristic frequency, meaning that a given neuron _res its action potential more likely if sound perceived by cochlea contains a frequency component close to its CF. When hair cells responsible for mechanoelectrical transduction of sound in cochlea die, neurons along the ascending auditory pathway lose their main source of excitatory input. In certain cases like prolonged exposure to a loud noise, age-related hearing loss, or blast exposure, a large portion of this cochlear input is lost causing disproportions in the neural excitation. Consequent homeostatic plasticity changes in the auditory cortex make neurons tune to previously weak non-cochlear inputs, yielding these neurons hypersensitive. Constant thalamocortical oscillation loop that feedbacks these hypersensitive neurons in both auditory thalamus and cortex then creates the sensation of tinnitus in the brain, according to our working model of tinnitus.

In the present study, we propose a novel approach to treat tinnitus patients with combined means of pharmaceutics and sound therapy. We base our treatment strategy on the previous decade-long research of our group that has been aimed to understand the fundamental properties and functions of the Cholecystokinin neuropeptide system in the brain. Cholecystokinin (CCK) is the most abundant neuropeptide in the central nervous system and many of its functions are still unclear. In the previous study, it was shown that direct local administration of the sulfated version of CCK octapeptide (CCK8s) through the injection cannula induced neural plasticity in the auditory cortex of rats. These findings were later supported in another study where CCK positive neurons were ontogenetically stimulated to induce long term potentiation (LTP) in the neurons of the auditory cortex. The treatment concept lies in strengthening the synaptic connection from remaining cochlear input towards deafferented thalamocortical neurons by meeting three core conditions for LTP:

. Presynaptic activation

. Postsynaptic activation

. Activation of CCKB receptors in postsynaptic neurons and triggering the molecular LTP-enabling pathway.

After such manipulation, auditory neurons would be rewired to still preserved cochlear input and their hypersensitivity would be presumably suppressed, leading to suppression of tinnitus.

The first major aim of this study is to confirm our hypothesis that intraperitoneal administration of CCK tetrapeptide (CCK4) followed by the sound stimulation can induce plastic changes in the auditory cortex similar to those that we have seen after direct manipulation of CCK positive neurons or local injection of CCK8s. CCK4 is a considerably smaller molecule than CCK8s and it was previously reported that contrary to CCK8s' blood-brain barrier impermeability (BBB) due to its size, CCK4 can indeed cross BBB and act on CCK receptors in the brain. Peripheral injection of CCK4, therefore, offers potential therapeutic possibilities towards our main goal. We performed electrophysiology experiments where we placed recording electrodes in layer IV of the mouse auditory cortex. After administering CCK4 and presenting sound pairing, we managed to potentiate excitatory postsynaptic potentials to previously weakly responding frequency band by a large margin after two ten-minute long pairing sessions. These results are in line with the previous findings of our group mentioned above. Importantly, this plastic change was target frequency-specific and did not seem to affect non-stimulated frequency bands.

After confirming the neuroplasticity capabilities of peripheral administration of CCK4 in neocortex, our next objective was to design behavioral models to induce and test tinnitus, followed by performing a long term behavioral tinnitus treatment study on mice. Mice were unilaterally exposed to very loud noise to induce chronic tinnitus. To qualitatively assess the tinnitus-like behavior, we designed our version of the Gap-Prepulse Inhibition of the Acoustic Startle Reflex (GPIAS) model. After animals were exposed to loud noise, their GPIAS performance significantly worsened. Notably, not only exposure frequency worsened, but also other bands. According to their tinnitus-like deficits profiles, we distributed mice into various treatment categories according to their badly performing frequency bands and performed Cholecystokinin-mediated sound therapy. Out of four treatment categories, all four showed significant improvement after CCK4 CMST sound therapy, compared to controls that showed no significant difference before vs after the treatment.

To confirm our GPIAS results we also adopted and improved the second behavioral model, the active avoidance Shuttle box. Animals that were not supposed to switch between two compartments during silent trials began to spontaneously switch after NIHL, indicating tinnitus-like behavior. After Cholecystokinin-mediated sound therapy, this behavior was significantly suppressed, further supporting our evidence that CMST can suppress tinnitus. No such effects were recorded in a control group.

We also provided additional supporting evidence, such as comprehensive distribution atlas of CCKA and CCKB receptors, Fluorescent CCK sensor data after peripheral administration of CCK4 and we investigated another behavioral model based on constant operant conditioning task in mice home cages.

Date of Award	11 Jan 2021
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Jufang HE (Supervisor)