Sexually differentiated calling patterns of Xenopus laevis are conveyed to the vocal organ by a dedicated neuromuscular system. Here, we define afferents to vocal motor neurons and determine whether the connectivity of the vocal pathway is sexually differentiated. The use of fluorescent dextran amines and the isolated brain preparation readily permitted identification of anterograde and retrograde connectivity patterns. The whole-mount preparation allowed us to observe projections in their entirety, including cells of origin of a projection (for retrograde projections), terminal fields (for anterograde connections), and fiber tracts. Major findings are the confirmation of a robust and reciprocal connection between cranial nucleus (n.) IX-X and the pretrigeminal nucleus of the dorsal tegmental area of the medulla (DTAM) as well as between DTAM and the ventral striatum (VS). Newly revealed is the extensive connectivity between the rostral subdivision of the dorsal nucleus raphe (rRpd) and candidate vocal nuclei. In contrast to previous results using peroxidase, we did not observe dramatic sex differences in connectivity, although some connections were less robust in female than in male brains. Some retrograde connections previously observed (e.g., anterior preoptic area to DTAM) were not confirmed. Plausible hypotheses are that a set of rhombencephalic neurons located in DTAM, the inferior reticular formation and n.IX-X are responsible for generating patterned vocal activity, that activity is modulated by neurons in rRpd, and that activity in VS (particularly that evoked by conspecific calls), together with effects of steroid hormones at many sites in the vocal circuit, contribute to the initiation of calling.

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