The cochlear nucleus (CN) is the first auditory structure to receive binaural information via CN-commissural connections. In spite of an abundance of evidence that CN-commissural neurons are glycinergic and thus inhibitory, physiological, and anatomical evidence suggests that a small group of CN-commissural neurons are excitatory. In this study, we examined the excitatory portion of the CN-commissural pathway by combining anterograde tract tracing with immunohistochemistry of vesicular glutamate transporters (VGLUTs) and retrograde tract tracing with immunohistochemistry of glycine and GABA. VGLUTs accumulate glutamate in synaptic vesicles and are prime markers for glutamatergic neurons. The terminal endings of CN-commissural projections were typically en passant or small terminal boutons, but large, irregular swellings were also observed, confined to the granule cell domain (GCD). Both small and large terminal endings in the GCD colabeled with VGLUT2, but not VGLUT1. In addition, some CN-commissural cells themselves received VGLUT2-positive puncta on their somata. After large injections into the CN, 37% of the total number of retrogradely labeled commissural neurons was immunonegative to glycine or GABA. Retrograde labeling after a restricted GCD injection revealed a majority of putative excitatory CN-commissural neurons as multipolar, in the marginal regions of the ventral CN, medially as well as in the small cell cap region and deep dorsal CN. These results provide direct anatomical evidence that an excitatory commissural projection is present, and VGLUT2 is associated with this pathway both as its source and as a recipient.
