Age-related hearing loss, presbycusis, is a complex disorder involving the interaction of both peripheral and central neurological deficits. Central auditory dysfunction may contribute to poor temporal discrimination of complex sounds such as speech. This research is timely since our population over 60 years old is increasing rapidly due to advances in medicine and nutrition as well as the advancing age of baby boomers. This study was designed to provide a better understanding of age-related changes in dorsal cochlear nucleus (DCN) physiology. DCN was chosen because it receives direct input from the auditory nerve and much is known about its neuronal morphology, physiology and circuitry. In young animals, DCN output neurons, fusiform cells, receive excitatory inputs from the acoustic nerve, which is modulated and shaped by inhibitory glycinergic inputs from nearby vertical cells. A number of studies in rodents suggested an age-related impairment of glycinergic neurotransmission. To access the functional impact of reduced putative glycinergic input in the central auditory system, this study compared the physiological responses of DCN neurons from young adult and aged rats in response set of simple and more complex acoustic stimuli. Single-unit extracellular recordings were made from two groups of DCN neurons: fusiform cells and cartwheel cells. Fusiform cells reflect the culmination of DCN processing, therefore were good candidates for studying the effect of aging on one ascending auditory stream. Two specific aims were directed at fusiform cell: SA1) Examine the effects of aging on fusiform cell response properties to simple tone burst stimuli; SA2) Examine the effect of aging on DCN output neuron response to complex temporal stimuli. A third aim, SA3) Examine the impact of aging on the response properties of cartwheel cells, a DCN inhibitory interneuron. Fusiform cells recorded from aged rats displayed significantly higher maximum discharge rates to characteristic frequency (CF) tones, fewer nonmonotonic CF rate-level functions and more wide-chopper type post-stimulus time histograms (PSTHs) when compared to neurons from young adult rats. These findings were consistent with an age-related loss of inhibitory glycinergic input. To elucidate how loss of inhibition could lead to functional deficits in temporal processing, fusiform cells were challenged to encode sinusoidally amplitude modulated (SAM) tones. DCN output neurons were presented with SAM tones at three modulation depths at 30 dB above hearing level/response threshold with the carrier frequency set to each unit’s CF. Temporal synchronicity to the SAM envelope was measured using vector strength from temporal modulation transfer functions (tMTFs). Firing rate to SAM tones was also assessed in rate modulation transfer functions (rMTFs). DCN output neurons from aged rats showed no loss of rate response (rMTF) but displayed a selective loss of temporal precision to SAM tones with significant age-related changes in peak vector strength (best modulation frequency), and the shape and category of tMTF. Wide-chopper PSTH types had significantly lower vector strength values than buildup and pauser PSTHs in both young and aged fusiform cells. Since a significantly greater proportion of aged neurons exhibited wide-chopper responses, this could explain, in part, the loss of temporal processing. The age-related response changes in the present study mimicked results from earlier studies were glycine inhibition onto young adult fusiform cells was pharmacologically blocked. Cartwheel cells receive excitatory inputs from granule cell parallel fibers as well as somatosensory dorsal column nucleus and project glycinergic inputs onto DCN output neurons. They appear to play a role in the integration of auditory and somatosensory inputs such as sensing head position. Aged cartwheel neurons exhibited signs of disinhibition showing increased spontaneous activity, increased maximum discharge rates and altered rate-level functions. The observed age-related changes in cartwheel cells are consistent with deafferentation studies using acoustic trauma. Collectively, the changes in DCN output neurons and cartwheel cells reflect a potentially maladaptive age-related neuroplasticity in response to a loss of excitatory acoustic nerve input. These in vivo extracellular findings were consistent with a global downregulation of glycinergic input within the DCN of aged rats. This reduced inhibition may contribute to functional deficits, particularly in activities that require precise timing of events such as response to speech-like stimuli.

FFifteen to thirty-five percent of the population in the United States experience tinnitus, a subjective "ringing in the ears". Up to 10% of tinnitus patients report their symptoms are severe and disabling. Tinnitus was induced in FBN rats using 116 dB (SPL) unilateral octave-band sound exposures centered at 16 kHz for one hour in an anesthetized preparation. Rats were assessed behaviorally by an operant conditioning paradigm as well as a gap detection method to verify the development of tinnitus. Both young (7 mos.) and aged (30 mos.) sound exposed rats showed significant elevated auditory brainstem-evoked response (ABR) thresholds for clix and all tested frequencies immediately after the sound exposure. Eighty days post-exposure, ABR thresholds for the young exposed rats were significantly close to the initial young control values while aged exposed rats showed residual thresholds shifts relative to aged controls. Sixteen weeks following sound exposure, young exposed rats showed significantly reduced gap detection at 24 and 32 kHz, suggestive of high frequency tinnitus. Aged exposed animals showed significant tinnitus-related behavioral changes near 10 kHz by both behavior methods. Message and protein levels of &alpha1-3 glycine receptor subunits (GlyRs), gephyrin, BDNF and its receptor TrkB were assessed in dorsal cochlear nucleus (DCN) fusiform cells 4 months post exposure utilizing quantitative in situ hybridization and immunocytochemistry. Young exposed rats showed significant decreases of GlyR &alpha1 protein at middle and high frequency regions in DCN unlike the contrasting increase of their message levels. Aged exposed rats showed higher &alpha1 subunit protein levels in the same high and middle DCN frequency regions. The GlyR anchoring protein, gephyrin, was significantly increased in both young and aged exposed rats, suggesting an intracellular receptor trafficking change following acoustic trauma. BDNF and TrkB were also increased over fusiform cells in both young and aged exposed rats. [3H] strychnine binding was used to evaluate DCN GlyR pharmacology and function following sound exposure. The age-related decrease in GlyR α1 protein was reflected in the significant age-related down-regulation of GlyR (Bmax). Tinnitus-related changes in GlyR &alpha1 protein level was reflected in the decline of the GlyR (Bmax) in young exposed rats and up-regulated GlyRs in aged exposed animals. The GlyRs in DCN of young exposed animals also demonstrated an increase in affinity, further suggesting a post-exposure receptor composition change. These findings suggest that both aging and/or sound exposure/tinnitus are associated with GlyR changes capable of altering alter the output of the DCN. Detailed characterization of these GlyR modifications could advance the development of novel selective drugs for tinnitus and age-related hearing loss.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-26T20:08:35Z No. of bitstreams: 1 ThawannMalfattiBorges_DISSERT.pdf: 27333324 bytes, checksum: 7928d876effa4fd0184f0b246ecd1c34 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-29T20:35:41Z (GMT) No. of bitstreams: 1 ThawannMalfattiBorges_DISSERT.pdf: 27333324 bytes, checksum: 7928d876effa4fd0184f0b246ecd1c34 (MD5) / Made available in DSpace on 2016-04-29T20:35:41Z (GMT). No. of bitstreams: 1 ThawannMalfattiBorges_DISSERT.pdf: 27333324 bytes, checksum: 7928d876effa4fd0184f0b246ecd1c34 (MD5) Previous issue date: 2015-06-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Tinnitus is the perception of a sound in the absence of a corresponding physical stimulus. It is not clear yet what mechanisms are involved in tinnitus and how it starts and/or becomes chronic. Due to the relationship between tinnitus and somatosensory trauma/stimuli, the dorsal cochlear nucleus (DCN), a region known to integrate somatosensory and auditory pathways, has been identified as a potential key structure in the generation of phantom sound perception. Here, we target specific neuronal populations in the DCN to allow further investigation on how this region may contribute to the generation of tinnitus signals that spread to other auditory areas. We examined the expression of optogenetic proteins (Channelrhodopsin 2 - ChR2; and enhanced Archaerhodopsin 3.0 - eArch3.0), targeting neurons expressing Calmoduline Kinase II alpha (CaMKIIa) promoter in wild-type C57/Bl6 mice and neurons expressing nicotinic acetylcholine receptor subunit alpha-2 promoter (ChRNA2) in ChRNA2- Cre transgenic C57/Bl6 mice, using local virus injection, verified by fluorescence microscopy. Unit responses were differentiated based on their electrophysiological response to sound. We then investigated if firing of neurons expressing optogenetic tools can be controlled in vivo and if the same neurons also fire action potentials in response to precisely timed sound stimulation. Both in vivo and preliminary in vitro data shows that neurons expressing ChR2 do respond to sound, and that they furthermore also can respond to light stimulation with a stable and similar waveform. Moreover, in vivo data shows that neurons expressing eArch3.0, responding to sound, will decrease their firing rate when exposed to green light. Thereby showing that optogenetic tools can be used functionally in the DCN, it is possible to further test tinnitus theories by, for example, producing an increased firing rate in the DCN, trying to mimic tinnitus; or inhibiting increased spontaneous firing rate in the DCN of animals with noise-induced or salycilate-induced tinnitus.

GABAB receptors play an important role in regulation of neuronal excitability and stability of neural microcircuits. It is well known that dysregulation of slow GABAergic signalisation can lead to many pathological conditions (epilepsy, anxiety etc.). Current research indicates that the imbalance in the inhibitory transfer, caused by changes in the expression of GABABR in the auditory system could play an important role in the progression of tinnitus. The goal of the present thesis was to determine the distribution of the GABAB receptor and its auxiliary subunit KCTD12 in the mouse auditory cortex and the dorsal cochlear nucleus (DCN). Furthermore, a change in GABAB receptor localization in the DCN was observed in mice exposed to an acoustic stress. The GABAB receptor was expressed across the entire auditory cortex, both on the body and on the neuronal fibres. On the contrary, KCTD12 was found only in a particular subgroup of neurons that includes VIP (vasoactive intestinal peptide) and cholecystokinin positive interneurons., GABABR and KCTD12 protein were found in all layers and in all studied cells types (fusiform, cartwheel and stellate) of the DCN. Acoustic trauma of the WT mice resulted in GABAB receptor internalization specifically in fusiform cells that are the main projection neurons of the...