Geng-Lin Li - US grants

DESCRIPTION (provided by applicant): Hair cells in the cochlea connect to auditory afferent fibers via ribbon synapses. Presynaptic graded potentials are converted here into postsynaptic spikes. The long-term objective of this study is to investigate the strategies for auditory signal encoding at this synapse. It has been suggested that hair cells tend to release more than one synaptic vesicle at a time (multivesicular release: MVR). However, the cellular mechanisms underlying MVR are poorly understood and its functional advantages are not known. Our first hypothesis is that MVR occurs when [Ca2+]j in hair cells crosses a threshold that triggers neighboring vesicles on a ribbon to pre-fuse with each other and release all their contents into the synaptic cleft simultaneously. We will determine the quantal response size (i.e., excitatory postsynaptic current (EPSC) amplitude evoked by a single vesicle fusion) and use this to quantify EPSC quantal content. We will find out if vesicles in MVR are from a single ribbon and investigate the Ca2+-dependence of MVR (i.e. determine its Ca2+ threshold). The second hypothesis concerns the function of MRV and has two parts. One part is that MVR can charge and discharge the membrane of afferent fibers more rapidly, helping them to fire spikes with higher temporal precision for phase-locking. The second part is that MRV provides a necessary varying factor on EPSC amplitudes evoked by repeated sinusoidal depolarizations of hair cells. This allows afferent fibers to avoid firing spikes at every sinusoidal cycle and the timing of spikes will not deteriorate due to spike refractory periods. We will measure EPSCs in response to a sinusoidal presynaptic depolarization and then simulate these EPSCs to either substitute MVR with evenly distributed single vesicle releases within a time window (e.g. 0.1 ms), or limit the variation of EPSC amplitudes within the variation of quantal responses (removing thus the variation in their quantal content). These simulated EPSCs will then be experimentally injected into afferent fibers under current-clamp to determine to what extent the phase-locking of spikes becomes deteriorated when MVR is absent. The third hypothesis to be tested is that fused synaptic vesicles can be recycled through fast endocytosis following MVR. We will use a 2-photon microscope to visualize FM1-43 dye loading to monitor vesicle recycling, and we will also make cell-attached capacitance measurements on hair cells to study vesicle recycling by monitoring capacitance changes with a time resolution of 50 ps or higher. RELEVANCE: In the United States, roughly 23,000 adults and 15,500 children have received cochlear implants, which restore part of their hearing by directly stimulating auditory nerve fibers with electrodes. However, the algorithms to stimulate the fibers according to the sound signal have been determined only empirically. The fundamental studies of afferent fiber spiking proposed here will provide guidance for significantly improving these algorithms, especially for adult patients whose auditory systems are fully developed and may thus have lost some of their plasticity and adaptability to different stimulus protocols.

Project Summary Auditory hair cells connect to afferent nerve fibers through ribbon synapses, where auditory signals are converted from graded membrane potentials in hair cells into spikes in afferent nerve fibers. In vivo studies conducted in adult animals have shown that in response to sound afferent fibers fire spikes with remarkable temporal precision, but the underlying synaptic mechanisms are still poorly understood. A major hurdle is the lack of a fully mature model system for dual patch-clamp analysis in vitro. The afferent fiber terminals in the adult mammalian cochlea are small and mostly inaccessible to patch-clamp analysis, and results from the immature cochlea are insufficient to explain in vivo findings. Here we propose to examine auditory transmission in the amphibian papilla of adult bullfrogs, which is a unique model system where spiking patterns of afferent fibers in vivo have been recapitulated with paired patch-clamp experiments in vitro. Using this model system, we will combine dual patch-clamp recording, glutamate uncaging and computer modeling to address a central and fundamental question in hearing: how small and fast-changing signals in auditory hair cells are transmitted to afferent fibers precisely and tirelessly. First, we will examine the desensitization properties of AMPA receptors in afferent fiber terminals, and determine how these properties allow the ribbon synapses to maintain synaptic strength under continuous glutamate release from hair cells. Second, we will demonstrate that Ca2+ influx caused by pre-depolarization leads to rapid priming of synaptic vesicles and makes them releasable in response to brief voltage changes. We will test the hypothesis that this priming of synaptic vesicles is mediated by a second Ca2+ sensor with biophysical properties distinct from the one for synaptic vesicle fusion. Lastly, we will elucidate the cellular mechanisms for multivesicular release, a hallmark of hair cell ribbon synapses. We will test three major candidate mechanisms: fusion pore flickering, nanodomain coupling of synaptic vesicles and Ca2+ channels, and coordination of synaptic ribbons. The results obtained through this project will answer several long-standing questions of inner ear physiology, and expand our knowledge of the fundamental rules governing synaptic transmission at auditory hair cell synapses.

Brain science will benefit from the capabilities in tracing complex animal behaviors down to ensembles of individual neurons, and moreover establishing a real-time closed-loop brain-interface, ideally with deep brain access in a free-moving animal. This research project aims to address the focus areas in this National Science Foundation program by merging novel neurotechnology, evaluation of neural circuits during performance of complex cognitive behaviors, and large-scale neuron ensemble analysis and closed-loop behavioral control. The outcome of this research will result in new technologies and computational tools that can be used across the field of neuroscience and behavior, strengthening research efforts of multiple research groups. The educational objectives of this proposal are aimed at training and inspiring young engineers and scientists who are equipped with the multidisciplinary background required to help define the future trajectory of brain interfaces and data sciences. The broader impacts of this project include: 1) advancing transformative device technologies for next-generation neurotechnology and providing new and more powerful tools for neuroscience studies, 2) educating underrepresented undergraduate and graduate researchers to contribute to the nation's workforce needs in biotechnology, 3) contributing to the K-12 science, technology, engineering, and mathematics education through weekend seminars and mentoring student-teacher pairs from local middle/high schools; and 4) promoting neuroscience and neurotechnology among local senior citizens and support groups for neurological diseases.

The research objective of this proposal is to combine high precision optoelectronic neural probes with real-time neural decoding to feedback optogenetic control over animal behavior. Such closed-loop neural interface will establish a generalizable technology platform to study complex animal behaviors using optogenetic tools and real-time learning. The proposed work will open ample research opportunities and form connections among hardware engineering, cognitive neuroscience, and data science. The intellectual merit of the proposed work will be evidenced by three major contributions: 1) demonstration of high-precision optogenetic brain interface that combines multiplexed recording from and bi-directional control over neuron ensembles, 2) demonstration of closed-loop brain interface that employs real-time neural decoding and adaptive learning to control animal behavior, and 3) characterization of complex decision-making using high-precision, multiplexed data linking multiple brain areas.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.