Shirin Soleimani
October 20th, 2017
Dr. Doyle
Scientific Communication – Annotated Bibliography
Farahbakhsh, N. A., & Narins, P. M. (2006). Slow motility in hair cells of the frog amphibian papilla: Ca 2+-dependent shape changes. Hearing Research, 212(1-2), 140-159. doi:10.1016/j.heares.2005.11.004
PURPOSE: To investigate intracellular processes that mediate the calcium/calmodulin (Ca2+/CaM)-dependent slow motility in low frequency auditory hair cells dissociated from one of the two auditory organs in frogs. Also to develop a method for quantitative analysis of shape change in isolated hair cells and devising a model for identifying different aspects of the responses to agents that modulate the geometrical attributes of these cells.
METHODS: Amphibian papillae were dissected out of decapitated leopard frogs. The hair cells were isolated and the recording chamber was analyzed under a microscope with various solutions. The hair cells where then perfused for 10-15 min with AP solution and then with inhibitors and ionomycin and finally with a hypo-osmotic solution, in order to record complete time course of shape change. Using DIC imaging system, the cell’s length, cross sectional area, average diameter and volume were monitored.
RESULTS: Varying intracellular calcium concentration can induce shape change in RAPHCs. The majority of the length change however involved an iso-volumetric length decrease. ML-7 applied alone did not induce a significant change in length or volume. The multifunctional Ca2+/CaM-dependent kinase inhibitors, KN-93 and KN-62 had effects similar to those of ML-7, which was that they did not express a volumetric shortening episode. Effects of MLCK and CaMK inhibitors are not additive, treatment with both showed similar results as treatment with each inhibitor alone. The MLCK inhibitor and the antagonists of Ca2+/CAM-dependent kinases impede the iso-volumetric shortening phase of the response to ionomycin.
In an attempt to identify additional steps in the calcium-calmodulin-dependent pathways that could contribute to the iso-volumetric shortening it was found that type 1 protein phosphatase inhibitors induce minor shortening on their own but do not significantly alter the phase 1 response. They appear to counter effects of the inhibitors of Ca2+/CAM-dependent kinases.
KEY RELEVANCE: Hair cells in non-mammalian vertebrates share many essential characteristics with mammalian counterparts but they lack an electromotile protein in their plasma membrane. Auditory hair cells in non-mammalian vertebrates provide a simple model to study the contribution to slow motility of intracellular pathways modulating the cytoskeletal structure, and thus hair cells’ geometrical shape, without the influence of voltage-dependent transmembrane motile elements. The experiments conducted suggest that slow motility has been proposed to provide some protection against acoustic overstimulation. It is induced by number of biochemical agents, such as Acetylcholine. These findings contribute useful information to the research of the hearing organ and provide a tool for more discoveries that concern hearing aspects, especially mechanisms that could potentially prevent hearing loss in several organisms.
Beurg, M., Nam, J. -., Crawford, A., & Fettiplace, R. (2008). The actions of calcium on hair bundle mechanics in mammalian cochlear hair cells. Biophysical Journal, 94(7), 2639-2653. doi:10.1529/biophysj.107.123257
PURPOSE: To examine and assess the origin of the fast adaptation and force generation mechanisms conducted by hair bundles. The results would record the time-dependent mechanics of mammalian cochlear hair bundles to answer the following questions: are the hair bundles influenced by MT channels? How does calcium influx through the channels affect their mechanics? What are the differences between inner hair cells and outer hair cells mechanics?
METHODS: The mechanical properties of hair bundles were measured by stimulation with calibrated flexible glass while simultaneously recording the mechanotransducer (MT) channels. This was done using a three-dimensional finite –element model which incorporated a kinetic scheme for MT channel gating and calcium binding. The displacement signal was measured at the peak of the MT current to assume an instantaneous stiffness and up to 1-3ms to calculate the “steady-state” stiffness. The force delivered was calculated from the difference between the displacement of the tip of the fiber and the piezoelectric actuator.
RESULTS: The force-displacement relationship at short times was approximately linear but at later times became increasingly nonlinear, with an increase in compliance that was time-dependent. The time course of the creep (which was the slower component) in the bundle motion happened synchronously with fast adaptation. For example, for the OHC, the mean time constant of the secondary component for small displacements was 0.48 ms, compared to 0.43 ms for the fast adaptation. In the IHC, the consequent time constants were 0.8 ms for the displacement and 0.55 ms for fast adaptation. Fast adaptation is originated by calcium arriving through open MT channels. It is predicted that a change in calcium influx would impact the bundle response to a force stimulus. It was observed that decreasing extracellular calcium concentration from 1.5 mM to 0.02 mM approximately doubled the stiffness. The effects of altering extracellular calcium were also seen on the channel release (b2) mechanism. For the MT current, the time constant of adaptation was slower (from 0.12 ms to > 1 ms) and the probability of channel opening at rest increased (from 0.07 to 0.26) as the internal calcium concentration when the channel was open (CFA) was lowered from 100 to 3 µM. In the channel release model, b2 is an important ingredient needed to predict the effects of calcium on hair bundle stiffness and the secondary component of bundle displacement that parallels fast adaptation in response to a force step. Although fast channel adaptation is primarily attributable to stabilization of the channel state (fCa) increasing the force needed to open the channel, the b2 process also accentuates fast adaptation.
KEY RELEVANCE: manipulating the calcium influx through the MT channels increases hair-bundle stiffness and reduces the nonlinearity in the force-displacement relationship. A nonlinearity relationship and a mechanical response synchronized with fast adaptation are essential to high frequency mechanical amplification in non-mammalian hair bundles. There was no significant difference in the mechanical properties of hair bundles between IHCs and OHCs These findings allow researchers to better understand the adaptation and amplification mechanisms of hair bundles in order to work towards figuring out ways to prevent or treat hearing loss in the future.
Chen, G. -. (2002). Effect of hypoxia on noise-induced auditory impairment. Hearing Research, 172(1-2), 186-195. doi:10.1016/S0378-5955(02)00582-8
PURPOSE: The purpose of this experiment is to determine the effects of hypoxia on permanent noise-induced hearing loss. Since oxygen tension in the cochlea is decreased during and following noise exposure, the noise-induced hearing loss could potentially be due to decreased blood flow.
METHODS: 63 Rats were exposed to 14.1 kHz noise at different intensities and hypoxic air – which was normal compressed air (20.9% O2) that was diluted with 100% nitrogen gas to oxygen concentrations of 18% and 10%. O2 – for 4 hours. Subjects were randomly divided into nine groups and cochlear compound action potential threshold loss and hair cell loss were measured after 4 weeks of exposure to those conditions. After 4 weeks, the cochlear compound action potential (CAP) thresholds were measured at frequencies of 2, 4, 6, 8, 12, 20, 24, 30, 35 and 40 kHz. Body temperature and temperature of the cochlea were maintained. Through a silver chloride electrode, the CAP signals were recorded and identified as a threshold.
RESULTS: A pilot experiment showed that rats (6 of them) exposed to 6% O2 died after 2-3 weeks in the chamber. The threshold of each group of O2 air was measured and it was found that CAP threshold in rats exposed to 10% O2 were slightly higher than the control group (20.9% O2). This difference was statistically insignificant, demonstrating that exposure to 10% O2 for 4.5 hours does not have a lasting effect on auditory functioning.
Even though the hypoxia did not result in hearing loss and hair cell loss, the added exposure to noise and hypoxic air together had an impact on hearing loss and hair cell loss than the noise alone. The NIHL potentiation by hypoxia showed to rise linearly with the hypoxia level. 10% O2 exposure considerably potentiated NIHL and 18% O2 exposure also exhibited an evident NIHL potentiation. This finding indicates that there is an increased risk of the auditory system to noise exposure when blood oxygen level is lower than normal.
KEY RELEVANCE: Noise induced hearing loss (NIHL) is the most common occupational disease in the United States. Oxygen-dependent processes in the cochlea may be necessary for protection and/or repair of damage caused by severe noise exposure. Therefore, hypoxia in the cochlea is considered as one of the mechanisms that potentially causes noise induced hearing loss. Since many people at risk of exposure to noise may also suffer from hypoxia, it is important for researchers to be aware of this factor in order to gain insight into specific causes leading to hearing loss and work towards providing preventative methods to avoid early onset of noise-induced hearing loss.
Tong, B., Hornak, A. J., Maison, S. F., Ohlemiller, K. K., Liberman, M. C., & Simmons, D. D. (2016). Oncomodulin, an EF-hand Ca2 + buffer, is critical for maintaining cochlear function in mice. Journal of Neuroscience, 36(5), 1631-1635. doi:10.1523/JNEUROSCI.3311-15.2016
PURPOSE: To assess the role of Oncomodulin (Ocm), which is a type of calcium binding protein, expressed predominately by in cochlear outer hair cells. Targeted deletion of Ocm caused progressive cochlear dysfunction.
METHODS: Seventeen kilobases of the Ocm gene containing exons 2-4 were cloned, engineered and sequenced. After a series of sequencing PCR and southern blotting, two Ocm heterozygote mutants were generated and crossed to create an Ocm homozygote mutant. The cochlea was then dissected and immunostained with antibodies to myosin VIIa and appropriate secondary antibodies. The cochleae were then embedded in a gelatin-agarose solution. After that, confocal z-stacks of specific frequency regions from each ear were obtained.
RESULTS: Deletion of exons 3 and 4 (which contain two Ca2+ binding motifs) and exon 2 (which contains N terminus of Ocm) demonstrated that the lack of Ca2+ buffer leads to progressive cohlear dysfunction starting after a period of one month. Ocm−/− mutants were greatly impaired at a young-adult age, with ABR thresholds 20–50 dB higher than those in wild-type mice. This corresponds to what would be considered a moderate-to-severe hearing loss in humans. Heterozygous mice show ABR thresholds similar to their wild-type counterparts. In Ocm−/− mutants, OHC loss was spotty, with alternating regions of intact and missing cells. While the results show that Ocm is not essential for development of cochlear function, it is essential for the protection of OHCs from damage in the mature ear.
KEY RELEVANCE: In hair cells, mobile EF-hand Ca2+-binding proteins or “buffers” may play a substantial role in Ca2+ homeostasis and signaling. The key findings of this experiment are important because targeted deletion of Ocm can now be related to cochlear dysfunction. In addition, since presence of Ocm protects hearing in the mature ear, it is important to develop mechanisms that prevent disruption of the Ocm gene in organisms.