Zhongmin Lu - US grants

Sound localization is a critically important part of sound detection for all vertebrates. Since vertebrates live in many different habitats, various species may use different cues to localize sound sources. Comparative auditory research will help establish a general biological context within which the hearing of all vertebrates, including humans, can be better understood. Sound localization usually refers to the processes of determination of the position of a sound source and is closely related to two fundamental questions: where does the sound come? and how far is the sound source? The proposed project will focus on the first question. This project is directed at understanding the ability and mechanisms of sound localization by a non-mammalian vertebrate, the fish Astronotus ocellatus. Directional hearing in fish has been studied since von Frisch and Dijkgraaf (1935). Previous behavioral, anatomical, and neurophysiological studies have demonstrated the ability of directional hearing in a few fish species and have give some ideas about possible mechanisms underlying sound localization. However, these data are very limited and are mainly obtained from two fish species: cod (Gadus morhua) and goldfish (Carassius auratus). The overall ability of directional hearing has not been systematically studied in any fish species, and what cues fish use to localize sound sources are still unclear. The proposed work will investigate directional hearing in Astronotus ocellatus using a cardiac conditioning method. The focus is on the following specific questions: 1) What is the directional sensitivity of fish in the horizontal, sagittal, and frontal planes? 2) What is the smallest angle that fish are able to distinguish between two spatially separated sound sources? 3) Are fish able to use the cue of phase difference between sound pressure and particle motion to discriminate sound sources from opposing directions?

DESCRIPTION: The goal of this study is to study directional hearing in fish, using the sleeper goby as the model. It has five aims: 1. Determine the structural polarity of sensory hair cells in each otolithic organ in 3D space using LEM and SEM. 2. Determine if individual end organs play differential roles in encoding sound direction along different planes. 3. Make structure-function relationships by dye-filling ganglion cells from which directional sensitivity had been assessed by intracellular recordings. 4. Identify any particular direction in which the fish is particularly sensitive and measure MAAs. 5. Determine the contribution of each end organ to the pattern of directional hearing.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of the PI's research is to understand neural mechanisms of sound localization in vertebrates, including humans. The primary goal of this proposal is to determine how the primitive authority system of fish functions in sound localization. The PI's previous studies and others have demonstrated that fish determine the axis at which a sound wave is propagating by using arrays of spatially oriented sensory hair cells in the ear. However, it is not known how the brain processes the peripherally coded directional information in order to extract the specific direction of the sound. The proposed work will elucidate mechanisms underlying central auditory processing of directional information. It has been proposed that the fish ear is stimulated by a sound wave through two distinct pathways: direct particle motion input and indirect pressure input via the swim bladder. It was hypothesized that fish (with a swim bladder) first determine the axis on which the sound is propagating and then determine its direction by comparing the timings of inputs carrying pressure and particle motion information within the brain. The PI will test the two steps of this hypothesis on two functionally distinguishable teleost fishes, the sleeper goby and goldfish. Specific aims address anatomical organization and functional processing of directional information in the medulla and midbrain: 1) Auditory nuclei responsible for directional coding. Experiments will be carried out to reveal central projection sites of the saccular, lagenar, and utricular nerves and medullary projection sites in the midbrain using both anterograde and retrograde labeling methods. 2) 3D structure and cytoarchitecture of the auditory nuclei. Combined with the neuronal tracing, boundaries of the auditory nuclei will be defined to create their three dimensional structures using Neurolucida. Golgi stain studies will characterize types, sizes, and orientations of the auditory neurons. 3) Brain representation of the peripheral map of directional coding. Using whole-cell filling of Neurobiotin and confocal imaging, a 3D map of peripheral directional coding will be reconstructed to reveal how the saccular map of directional coding is represented in the medulla. 4) Directional response properties of auditory medullary and midbrain neurons. Single-cell recording and filling will characterize directional response properties, locations, and morphologies of auditory medullary and midbrain neurons to form a neural map(s) of best response axes of the neurons. 5) Differential-phase sensitive neurons. Single-cell recording will determine if there are neurons in the medulla and/or midbrain of the goldfish that encode specific timing differences between particle motion and pressure inputs

DESCRIPTION (provided by applicant): The zebrafish (Danio rerio) has become a unique vertebrate model for human disease research in hearing and balance because of a combination of powerful genetics, excellent embryology, and exceptional in vivo visualization in one organism. Recent studies use forward and reverse genetic manipulations and developmental approaches to generate a large variety of zebrafish hearing mutants in order to identify novel deafness genes and investigate auditory functions of target genes. However, auditory abilities of larval zebrafish are unknown due to lack of a method that can evaluate hearing function of zebrafish at early stages. This project has two specific aims. First, the PI will develop a reliable method that is suitable for determining auditory abilities of zebrafish larvae. There are two general ways (loudspeaker and shaker table) to produce acoustic stimuli that can activate sensory hair cells in the inner ear of fish. There are two common methods (auditory evoked potential recording and behavioral classical conditioning) to assess the auditory function of fish. The PI will investigate what setup and method are best suitable for determining auditory thresholds of young larval zebrafish. Results of this project will provide the zebrafish research community a hearing test that can precisely assess hearing defects of zebrafish mutants used for studying auditory disorders in humans. Second, the PI will investigate how the auditory function of wild-type zebrafish enhances during the early development after fertilization. The timeline of development of best frequency, best sensitivity, and audible frequency range will be determined for zebrafish of several different age groups from just hatching to adulthood. The PI will determine the critical period of development of hearing function and identify the time when the auditory capacities reach a plateau. We know little about what young larval zebrafish can hear and how their auditory function develops. Results from this study will fill this knowledge gap to further establish the zebrafish as a model system for auditory research. Easy accessibility of zebrafish embryos right after fertilization makes zebrafish an ideal candidate to investigate the early hearing development in vertebrates. Results from this study will help us better understand evolution of the early development of auditory function in vertebrates including humans. The method developed by this project will enable the zebrafish research community to precisely assess auditory functional defects of zebrafish mutants used for studying human hearing disorders and help further establish the zebrafish as a vertebrate model system for human hearing research. Results from this study will fill the knowledge gap of auditory abilities of larval zebrafish and provide insight into evolution of the early development of auditory function in vertebrates including humans.