Sound localization critically relies on brainstem neurons that compare information from the two ears. The conventional role of the lateral superior olive (LSO) is extraction of intensity differences; however, it is increasingly clear that relative timing, especially of transients, is also an important function. Cellular diversity within the LSO that is not well understood may underlie its multiple roles. There are glycinergic inhibitory and glutamatergic excitatory principal neurons in the LSO, however, there is some disagreement regarding their relative distribution and projection pattern. Here we employ in situ hybridization to definitively identify transmitter types combined with retrograde labeling of projections to the inferior colliculus (IC) to address these questions. Excitatory LSO neurons were more numerous (76%) than inhibitory ones. A smaller proportion of inhibitory neurons were IC‐projecting (45% vs. 64% for excitatory) suggesting that inhibitory LSO neurons may have more projections to other regions such the lateral lemniscus or more distributed IC projections. Inhibitory LSO neurons almost exclusively projected ipsilaterally making up a sizeable proportion (41%) of the transmitter type‐labeled ipsilateral IC projection from LSO and exhibited a moderate low frequency bias (10% difference H‐L). Two thirds of excitatory neurons projected contralaterally and had a slight high frequency bias (4%). One third of excitatory LSO neurons projected ipsilaterally to the IC and these cells were strongly biased toward the low frequency limb of the LSO (37%). This projection appears to be species specific in animals with good low frequency hearing suggesting that it may be a specialization for such ability.

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