Figure 4.

Photomicrographs of labeled CNA neurons. (A) Retrogradely-labeled (CTB+) neurons containing black CTB granules following single immunostaining for the presence of the retrograde tracer in the projection neurons (arrowheads). The CTB+ neuron in the bracket of A is further illustrated in the inset (B), at higher magnification, to demonstrate the presence of dense aggregates of black CTB granules within its cytoplasm. (C) Another example is presented of a CTB+ neuron with single labeling showing clear punctate black granules that are present not only within the soma, but which also fill the cell processes of this CNA neuron that projects to the NPO. (D) Double-labeling studies showing a glutamatergic neuron that projects to the NPO which contains black CTB granules with brown (VGLUT2)-immunostained cytoplasm (arrow). Brown-stained neurons, that were single-labeled for VGLUT2 (arrowheads), were present near this double-labeled neuron (arrow). The inset (E) depicts another type of single-labeled neuron (from a separate section) that contains black CTB granules without brown-stained VGLUT2 immunoreactivity in its cytoplasm. Brain sections of A to C were counter-stained with Pyronin-Y. Abbreviations: CNA, central nucleus of the amygdala; CTB, cholera toxin B-subunit; NPO, nucleus pontis oralis; VGLUT2, vesicular glutamate transporter 2.