Major Research Interests
Overview of research interests
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Concepts |
Models and Techniques |
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Role of neural activity pattern in development of hair cell innervation/synaptic structure |
Transgenic mice expressing altered post-synaptic neurotransmitter receptor function- genetic nulls, over-expressers, and point mutants altering receptor kinetics; confocal light microscopy and electron microscopy; auditory system physiology, neural tract tracing |
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Molecular mechanisms important in establishing cochlear sensitivity/afferent functionality |
Transgenic mice, peripheral auditory system physiology |
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Endogenous cellular signaling systems involved in protection against both sound and drug-induced damage |
Targeted protein expression analysis via standard western blot techniques; global proteome analysis via liquid chromatography tandem mass spectrometry (iTRAQ and SILAC LC-MS/MS); in vitro and in vivo models |
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Gene and protein expression profiling of normal and disrupted development leading to altered synaptic structure and function in the cochlea |
Normal and transgenic mice, Affymetrix-based gene expression profiling, iTRAQ labeling and LC-MS/MS, bioinformatic (statistical) analysis |
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Role of peripheral auditory activity in refining central auditory system tonotopy, synapse formation and function |
Transgenic mice exhibiting altered efferent feedback to the cochlea, delineation of tonotopic fields, Affymetrix-based gene chip analysis |
I. Current research interests and ongoing work
My work investigates mechanisms (gene expression and cell signaling cascades) by which descending neural input to hair cells and local peptide based paracrine signaling regulates synaptic structure and strength of both this efferent synapse as well as the afferent synapse and associated central neural circuitry. The immediate goals of my current research are three-fold. First, I seek to provide a framework useful for understanding the mechanisms by which synapse formation occurs in the auditory periphery. This determines the nature of the initial information flow to the CNS. Second, I seek to define the molecular mechanisms by which hearing sensitivity and susceptibility to noise-induced and ototoxin-induced hearing loss is set and modulated. While it is well recognized that sensitivity can be modulated over a timeframe of seconds, I hypothesize that this can also occur over the course of hours to days, and function in a proactive, protective manner to shield hair cells from damage. Finally, I ask how normal and abnormal peripheral states feed into CNS auditory systems during development to modulate synaptic (structural and functional) plasticity. Defects in CNS processing can alter sub-cortical based abilities such as localization of sound in space, to higher-level cognitive abilities of speech perception and disease states such as tinnitus. My work toward these goals can be split into two complementary trajectories. First, during my time as a postdoctoral fellow and later as a Staff Scientist at the Salk Institute and most recently as an Assistant Prof. at Tufts, my group has discovered that nicotinic acetylcholine receptor (nAChR) activity modifies CNS derived neural innervation and synaptic terminal structure in the cochlea, as well as the mechanisms by which this modulation is established. We have also used the Affymetrix gene chip-based system to define the changes to global gene expression following manipulations of the hair cell expressed nAChRs and found a delayed developmental state of the cochlea following silencing of nAChR activity. Secondly, we have discovered a local signaling system within the cochlea that is molecularly equivalent to the classic hypothalamic-pituitary-adrenal axis stress-response system composed of corticotropin-releasing factor (CRF) and its receptors, along with POMC, ACTH, and the ACTH receptor MC2R. We have elucidated how CRF and urocortin, a CRF-related peptide, and their receptors participate in setting basal hearing sensitivity, and how the system modulates susceptibility to acoustically mediated trauma. In a related line of work, we have begun to use quantitative mass spectrometry (specifically iTRAQ labeling) to examine the proteomic response of inner ear derived cells following presentation of ototoxic drugs. Each research line is briefly presented below.
The role of nAChR genes in the development and function of efferent synapses with hair cells- We use genetically manipulated mice as tools to investigate questions of interest pertaining to inner ear neurobiology. Our most mature line of research uses these tools to investigate the role of nAChR activity in modulating synapse formation and function in the cochlea. Our results demonstrate that prior to hearing onset, nAChR activity modulates the structural state of the efferent presynaptic terminal by altering cell adhesion molecule expression via perturbation of CREB signaling pathways. We have further shown that a bi-directional signaling system between hair cells and efferent synaptic terminals is responsible for establishing and maintaining normal presynaptic structure and function (Murthy et al., 2009). In short, we have revealed novel roles for the olivocochlear (descending efferent) system beyond that understood for adult hearing, which typically modeled the system as one of providing neural feedback that altered the mechanical state of the inner ear. Our work opens possibilities for a deeper understanding of cholinergic synapse formation in general terms, a topic not well addressed outside of the neuromuscular junction. We have also recently published a global transcript expression study using standard Affymetrix gene chip analysis over specific postnatal developmental stages to reveal the global changes in gene expression not only during normal inner ear development, but also how that development is altered in the face of genetic null ablation of the alpha9 nAChRs expressed by hair cells (Turcan et al., 2010). Our work has shown that significant changes occur in the expression of genes involved not only in the efferent system as we expected, but unexpectedly also in the afferent signaling pathway into the brain. That afferent function can be modified by abnormal efferent function was made even more clear with results demonstrating auditory-induced seizures in mice carrying a point mutation in the alpha9 gene that significantly alters the desensitization kinetics of the receptor. As a result of our work in modifying olivocochlear function via induced genetic manipulations, it has become increasingly clear that abnormal early olivocochlear activity alters the normal pre-hearing activity of the cochlea, resulting in altered central neural activity and circuitry development, demonstrating that modulation of early, pre-hearing cochlear activity is involved in synaptic refinement of CNS auditory nuclei. This probably reflects similar mechanisms at play in the ear as those demonstrated previously in the visual system.
The role of corticotropin releasing hormone and associated receptors in cochlear development and processing- While the olivocochlear system modulates auditory thresholds over the span of seconds to perhaps minutes, I hypothesized that the cochlea may also express a system through which longer lasting modifications to sensitivity may be elicited. This interest and the work that has evolved from these ideas began as a collaboration with Dr. Wylie Vale and Kuo-Fen Lee while I was a Staff Scientist at the Salk Institute. My work described the expression of the corticotropin releasing hormone related peptide urocortin in the cochlea, as well as the functional consequences resulting from ablation of the urocortin gene (Vetter et al., 2002). This work was the first to describe the expression of both a CRF-related peptide and the two CRF receptors in the cochlea. Results also demonstrated a role for urocortin in setting auditory sensitivity. Since moving to Tufts, my lab has also recently uncovered a system of peptides and G-protein coupled receptors expressed in the cochlea that are also part the well-known HPA axis. This work has recently been submitted for publication. The cochlear HPA-equivalent signaling system is wholly localized in the inner ear, and we hypothesize that it plays a role in modulating inner ear sensitivity over longer periods of time (minutes to hours). Supporting this hypothesis, we have gone on to use genetically manipulated mice carrying deletions of the two main CRF receptors to elucidate the mechanisms by which the CRF system functions in the inner ear. Our results demonstrate that activation of these receptors controls the sensitivity of the inner ear via coordinated affects on trafficking and expression of glutamate receptors, ATP receptors, and connexin subunits. Mutant mice lacking CRFR1 have decreased hearing sensitivity in addition to significant neural innervation defects to the hair cells (this work has recently been submitted). Interestingly, null ablation of CRFR2 results in a significant increase in hearing sensitivity, and a significantly greater susceptibility to noise-induced hearing loss occurs (Graham et al., 2010), demonstrating the extremely narrow operating point at which the inner ear normally functions in balancing sensitivity with susceptibility to acoustic injury. Thus far, work on the cochlear CRF system suggests that it serves to establish a balance of synaptic tone in the afferent auditory pathway. I next reasoned that if the CRF system is involved in setting sensitivity such that it is balanced with susceptibility to noise-induced hearing loss, it might also represent an endogenous system involved in general protection against various cellular stress events. Using a combination of standard biochemical assays and a mass spectrometry-based differential proteomics approach (Vetter et al., 2009), we have gone on to explore CRFR2 signaling in an in vitro model of cochlear derived cells to better define the changes that take place during drug-induced damage to cells of the inner ear. We showed that pre-activation of the cells with a CRFR2 specific agonist protects the cells from ROS generation, as well as superoxide dismutase and caspase 3 activity. Following an in vitro proteomic analysis employing quantitative differential mass spectrometric procedures and iTRAQ labeling of the cells, we further detailed numerous differentially expressed proteins, many of which were not previously linked to cochlear function before (Basappa et al., 2010). Thus, the CRH system seems ripe for exploitation to generate novel interventional strategies against stress-related insults to the inner ear that result in hearing loss.
While my main research focus includes the inner ear, we address many basic neuroscience problems from a cell and molecular biology approach that are related to the role specific genes and proteins play in cell signaling and plasticity of neurotransmission, neuronal circuitry development, and pathological states. We are also poised to move aspects of our work back to investigating central auditory system function in some of our mutant lines, especially concerning the impact of early neural activity on the development and function of central auditory circuitry. I believe one of the strengths of our lab is that we integrate numerous techniques from classical morphological and physiological analyses to cutting edge molecular and genetic/proteomic techniques to address problems of interest.
Turcan S, Slonim DK, Vetter DE (2010) Lack of nAChR activity depresses cochlear maturation and up-regulates GABA system components: Temporal profiling of gene expression in a9 null mice. PLoS One 5:e9058. doi: 9010.1371/journal.pone.0009058.
Vetter DE, Katz E, Maison SF, Taranda J, Turcan S, Ballestero J, Liberman MC, Elgoyhen AB, Boulter J (2007) The a10 nicotinic acetylcholine receptor subunit is required for normal synaptic function and integrity of the olivocochlear system. Proc Natl Acad Sci U S A 104:20594-20599.