Thus, SGN axons are arrayed in a high-to-low-frequency tonotopic map along the dorsoventral axis of the CN (Young and Oertel, 2004). Similar tonotopy is observed in CN neuronal responses themselves, determined both electrophysiologically (Luo et al., 2009) and by Fos induction ( Friauf, 1992; Saint Marie et al., 1999). We injected FosTRAP mice with 4-OHT during a 4 or 16 kHz continuous pure tone stimulus to TRAP CN neurons tuned to those frequencies. To increase the total number of TRAPed cells, we took advantage of TRAP’s ability to integrate IEG expression over

time by using a 4 hr pure tone stimulus during the TRAPing period. Then, 4–5 days later, we delivered a second 4 or 16 kHz stimulus for 1 hr, sacrificed the mice 1 hr learn more later, and processed the tissue for Fos immunostaining (Figure 5A). Thus, TRAPed cells represent neurons Wnt inhibitor activated by the first stimulus, and Fos protein immunopositive (Fos+) cells represent neurons activated by the second stimulus. Consistent with prior results, we found that 4 kHz stimulation during the second epoch induced Fos expression in clusters of cells in all three CN

subdivisions that were located more ventrally than the clusters that were Fos+ after 16 kHz stimulation. Similar results were observed for TRAPed cells. When the tone frequency was the same for the two stimulus epochs, the TRAPed and Fos+ populations overlapped, and the 4 kHz cluster was localized more ventrally than the 16 kHz cluster (Figure 5B, first and third columns). Within mice receiving stimuli of two different frequencies, the cells TRAPed by the 16 kHz stimulus Ketanserin were dorsal to Fos+ cells induced by the 4

kHz stimulus (Figure 5B, second column), whereas the reverse was true when the 4 kHz stimulus was TRAPed and the 16 kHz representation was revealed by Fos immunostaining (Figure 5B, last column). These qualitative impressions were confirmed by the quantification of the numbers of TRAPed and Fos+ cells in bins spanning the dorsoventral axis of the central dorsal cochlear nucleus (DCN; Figure 5C). In general, the populations of TRAPed cells were less sharply confined along the dorsoventral axis than the population of Fos+ cells. This may reflect the longer stimulus used for TRAPing (4 hr, versus 1 hr for Fos immunostaining) or some general noise in the TRAP approach. Regardless, this analysis supports the observations from individual sections that both TRAP and Fos immunostaining reveal similar tonotopic maps along the dorsoventral axis of the DCN. We also quantified the overlap between TRAPed and Fos+ cells for the different treatment groups across the entire extent of the DCN.