3D model of frequency representation in the cochlear nucleus of the CBA/J mouse.

Hearing begins in the cochlea, where sounds of different frequencies stimulate hair cell receptors at different positions along the basilar membrane. This mapping is approximately exponential with distance, with a low-to-high frequency gradient established from the apex to the base, respectively (von Bekeesy, 1960; Liberman, 1982). Stimulated hair cells, in turn, activate auditory nerve (AN) fibers with which they are connected, relaying information from the periphery to the central nervous system (reviewed by Nayagam et al., 2011). Because the activity of AN fibers reflects their cochlear point of origin, there is a place-frequency map in the cochlea (Liberman, 1982). Early anatomists recognized that the AN projects into the cochlear nucleus (CN) following a stereotyped plan (Ramon y Cajal, 1909; Lorente de No, 1933). Fibers tuned to low best frequencies (BFs) are distributed at more ventral locations throughout the CN, and fibers with progressively higher BFs are distributed progressively dorsally (Fekete et al., 1984; Ryugo and May, 1993). This arrangement corroborates physiological observations of frequency tuning (Rose et al., 1959; Bourk et al., 1981; Spirou et al., 1993), suggesting that tonotopy in the CN is due to the spatial pattern of synaptic connections made by auditory nerve fibers on CN neurons (Ramon y Cajal, 1909; Lorente de No, 1933; Fekete et al., 1984; Ryugo and May, 1993). Studies of tonotopy in the CN have largely been reduced to 1- or 2D, with few studies providing quantitative results in 3D space (Bourk et al., 1981; Muller, 1990; Luo et al., 2009). A complete model of frequency representation in the CN would be beneficial for evaluating possible frequency specializations in the animal or pathologic changes in tuning. Among the scant data currently available, there also exists no straightforward method for experimenters to freely view and interpret spatial data. Atlases of auditory structures have typically consisted of sets of standardized serial sections to which experimental sections must be matched for interpretation (e.g., Kiang et al., 1975; Willard and Ryugo, 1983; Franklin and Paxinos, 1997; Trettel and Morest, 2001; Cant and Benson, 2005). Such methods are approximate and highly subjective, requiring the experimenter to make assumptions about the angle at which their tissue was sectioned and relative similarity of an experimental section to a given atlas section. The mouse has emerged as a model for mammalian auditory research (Willott, 1983, 2001; Steel and Kros, 2001; Liu, 2006). A number of studies have characterized aspects of frequency representation within the mouse cochlea (Ehret, 1975; Ou et al., 2000; Muller et al., 2005) or CN (Ryugo et al., 1981; Ehret and Fischer, 1991; Berglund and Brown, 1994; Luo et al., 2009), but none have provided the kind of detail that would enable a complete quantitative and comparative assessment of frequency representation in 3D. To this end, we adapted techniques used in insect neuroanatomy (Rein et al., 2002; Jenett et al., 2006) to construct a 3D template of the CBA/J mouse CN. Next we analyzed the trajectory of physiologically characterized labeled auditory nerve fibers resulting from injections in the CN and mapped each case to our 3D template using automated alignment algorithms. Finally, we used this composite dataset to develop a quantitative map of tonotopy in the CN of this mouse strain. This type of organization has been explored in other species (Feng and Vater, 1985; Muller, 1990), but we have expanded on past methods to allow us to generalize our findings across a number of experimental cases and to create an interactive model. This model can serve as a tool for visualizing CN tonotopy (e.g., virtual arbitrary slices) and a quantitative reference upon which hypotheses concerning frequency and location in the CN can be tested.