- Stochastic resonance in receptor cells and excitable cells
- Signal amplification in receptor cells
- Stochastic approaches to study molecular motors in the hair bundle
- The machinery and dynamics of negative feedback loops in the cell, especially for adaptation
- The role of spontaneous synaptic activity in development
- The development of the optokinetic response in zebrafish
1-Adaptation motors in hair cells.
Sensory systems adapt and desensitize, that is, our senses’ response to a maintained stimulus declines with time (a blessing with bad odors, a curse with good wines). We studied the tiny myosin motors which in hair cells constantly re-adjust the cell’s sensitivity to mechanical forces. To do this we activated the motors externally by applying the transduction channel blocker gentamicin to hair bundles at rest. Gentamicin reduces the entry of Ca++ through transduction channels, and the consequent reduction in intracellular Ca++ is similar to that occurring when transduction channels close. The adaptation machinery “interprets” this Ca++ reduction as a sign that the channels have closed, and in response it attempts to compensate by activating the motors that stretch the tip links responsible for chanel opening. We expected that, as the motors became activated, and as the hair bundle would (see displacement trace below) the variance (random movement) of the hair bundle’s position would increase. We observed an increase in noise (see the variance in figure below) whose amplitude and temporal characteristics are consistent with the behavior of myosin, a family of molecular motors, observed in other systems.
2-Stochastic Resonance in Mechanoelectrical Transduction.
Because hair bundles in inner hair cells of the mammalian cochlea are not restrained, they undergo significant Brownian motion, a characteristic traditionally thought to blunt the sensitivity of hearing. Our work suggests that Brownian motion of the hair bundle serves to enhance the sensitivity of mechanoelectrical transduction. The figure below shows how the signal-to-noise ratio (SNR) of mechanoelectrical transduction in two different hair cells improves (up to a point) with the addition of mechanical noise to the hair bundle. The optimal noise level is similar to that of Brownian motion in an unrestrained bundle.
3-Calcium Dynamics in Hair Cells.
After leaving the Hudspeth lab I took a tenure-track job in the department of physiology at the Emory University School of Medicine. My first project studied the dynamics of calcium in hair cells. Mobile Ca2+ buffers in hair cells have been postulated to play a dual role. On one hand, they carry incoming Ca2+ away from synaptic areas, allowing synapses to be rapidly reset. On the other hand, they limit the spread of free Ca2+ into the cell, preventing cross-talk between different pathways that employ Ca2+ as a second messenger. We obtained evidence for such mobile Ca2+ buffers in hair cells by comparing the patterns of Ca2+-induced fluorescence under whole-cell and perforated-patch recording conditions. Fluorescent signals under perforated-patch conditions (where the cytosol Ca2+ buffers are maintained) are relatively weak and limited to the immediate vicinity of the membrane (see figure) where they appear as narrow peaks. These observations can be explained by a model that incorporates endogenous fixed and mobile Ca2+ buffers. Our experiments also suggested that the mobility of the endogenous buffer might be higher than previously thought. A high buffer mobility is expected to enhance the cell’s ability to rapidly modulate transmitter release.
Two calcium microdomains (fluorescence peaks) on opposite sides of an isolated hair cell.
4-Postdoctoral Research: The Biophysics of Mechano-electrical Transduction in Hair Cells
- My first goal at the Hudspeth lab was to localize the site of transduction in hair cells, the mechanosensory receptors of the vestibular and auditory systems of vertebrates which is crucial for understanding how transduction channels respond to mechanical forces. Having failed at answering this question using calcium imaging, I took advantage of the sub-micrometer spatial resolution afforded by iontophoresis to localize the site of transduction in hair cells from the bullfrog’s sacculus. In these experiments I recorded transduction currents from isolated hair cells using the whole-cell recording technique while an iontophoretic electrode filled with aminoglycosides was used to focally inactivate transduction currents. The mechano-electrical transduction current was maximally blocked when the iontophoretic electrode was located near the tips of stereooclia, indicating that it is here that the transduction channels are located.
- I then focused on the forces exerted by the gating springs in hair bundles. I helped to develop a displacement-clamp system to study the forces that arise during mechanoelectrical adaptation. This system enabled me to rapidly displace individual hair bundles while measuring the force required to hold them in place while they underwent adaptation. I also successfully employed the displacement clamp to measure the forces exerted at rest by the gating springs. This question can be approached because the gating mechanism is reciprocal: not only does the position of the bundle determine the tension of the gating springs, but changes in the springs’ tension are communicated to the hair bundle. These experiments showed that individual gating springs exert a force of about 57 pN at rest. In agreement with previous evidence, I also observed that the gating springs account for at least one third of the bundle’s dynamic stiffness. In a parallel series of experiments I used gentamicin to measure the force, above that present at rest, that the gating springs are capable of exerting. These experiments were based on previous observations which indicated that the tension level in the gating springs is determined by the entry of Ca into the hair cell through the transduction channels. By blocking the Ca entry that takes place at rest I was able to drive the gating springs to exert an additional 29 pN of force beyond that present at rest. These results indicate, after taking into account the number of gating springs and the geometrical gain, that the motors that determine the tension of individual gating springs can exert a force of at least 12 pN. Several myosin-like molecules, acting along the stereociliary actin cytoskeleton could account for a force of this magnitude. These data also provide an independent estimate of the single channel gating force of about 100 fN
- Finally, in collaboration with Drs. A. J. Hudspeth and Vladislav Markin, I investigated the origin of two-tone distortion products. These are auditory illusions, known for several centuries but of underdetermined origin. Our experiments in solitary hair cells suggested that they could be due to the nonlinear mechanical properties of the transduction apparatus. Later work in the laboratory of Dr. Mary Ann Cheatham at Northwestern University has provided further support for this hypothesis.
5-Dissertation Research: Kinetic and Physiological Differences Between Embryonic- and Adult-Type Acetylcholine Receptors in Rat Developing Muscles
- My graduate work concerned the development of the neuromuscular synapse. I studied the changes in the properties of the acetylcholine receptor (AChR) channel that take place during the first few weeks after birth in rats. My graduate research focused on two questions related to this developmental process. First, how does channel gating differ between embryonic- and adult-type AChRs? To compare the gating behavior of embryonic- and adult-type AChRs, I recorded from cultured rat myotubes using the patch-clamp technique. AChRs were activated with ACh and with suberyldicholine. These single-channel recordings indicated that both types of receptor enter at least two distinct open states, three distinct closed states, and one subconductance state. This result suggests that the two channel types are gated by similar mechanisms. The shorter apparent open time of the adult-type receptor is due to a larger closing rate. In addition, I studied the kinetic properties of both types of AChRs by analyzing the rising phase of miniature endplate currents in intact muscle fibers. These experiments provided information that confirmed my observations in cultured myotubes.
The arrow head points an endplate in a living soleus muscle fiber in vitro.
- The second issue that I addressed in my doctoral research was that of the consequences for neuromuscular transmission derived from the expression of different forms of the AChR. Experiments carried out by S. Vicini in Dr. Stephen Schuetze’s lab, had previously suggested that the frequent spontaneous contractions that are observed in developing muscle fibers are caused by spontaneously occurring miniature endplate potentials (MEPPs). To establish the extent to which these contractions are due to the presence of the embryonic forms of the AChR, I decided to inject (electrically) previously recorded miniature endplate currents (MEPCs) of various amplitudes and timecourses into neonatal muscle fibers, and to record the ensuing depolarization with a second, independent microelectrode. Neonatal fibers responded similarly to spontaneous and to injected MEPCs. The injection of embryonic- and adult-type MEPCs induced depolarizations of approximately the same amplitude, indicating that the larger amplitudes of neonatal MEPPs are not due to the long duration of neonatal MEPCs. Embryonic-type MEPCs proved to be far more effective than the adult type in exciting neonatal muscle fibers. Because both embryonic and adult MEPPs have similar amplitudes, I attributed this higher efficiency to the long duration of embryonic MEPCs and therefore to the presence at the endplate of the embryonic form of the AChR. Contractile activity is necessary for the differentiation of both the synapse and the muscle fiber. These experiments demonstrated that the embryonic form of the AChR is essential for spontaneous contractile activity, and therefore they provided the first evidence for the functional significance underlying this developmental process.