Figure 4. Representative example of LHRH neurons expressing the immediate-early gene (IEG) Fos during the afternoon of proestrus, which project to the median eminence. The retrograde tracer Fast-blue (0.2%), diluted in sterile saline, was administered in the femoral vein of cycling female rats. Four days later, the animals were deeply anesthetized and rapidly perfused with 4% paraformaldehyde between 1730 and 1800 h of proestrus afternoon. The frozen brains were mounted on a microtome and cut into 30-mm coronal slices. Panel A shows a clear example of 5 LHRH-immunoreactive neurons that contain the retrograde tracer Fast-blue (panel B) and the nuclear Fos protein (panel C). Although these data indicate that LHRH neurons expressing Fos during proestrus project to the median eminence (see filled arrowheads), we have also observed that some LHRH neurons do not contain both the Fast-blue and the Fos-immunoreactive protein. Indeed, some LHRH neurons expressed Fos without containing the retrograde tracer and vice versa. LHRH neurons were stained by means of immunocytochemistry technique using a fluorescein (FITC)-labeled second antibody, while Fos proteins were stained using a rhodamine-labeled antibody. Small arrowheads show LHRH-immunoreactive neurons (panel A) that contain the retrograde tracer Fast-blue (panel B) and the nuclear Fos protein (panel C). (Taken from Rivest and Rivier, ref. 229.)