Application of a fluorescent dye to study connectivity between third ventricular preoptic area grafts and host hypothalamus.

Abstract

The mutant hypogonadal (hpg) mouse lacks a functioning gene for the neurohormone gonadotropin releasing hormone (GnRH). Previous studies from our laboratory had indicated that the initiation and maintenance of reproductive function in these mice could be brought about by the implantation of normal fetal grafts into adult hosts. Testicular or ovarian growth and other indicators of normal neurosecretory output were always accompanied by survival of GnRH neurons and growth of GnRH axons into the host median eminence where such axons terminate on the hypophysial portal capillaries. To determine if other connections exist between graft and the host hypothalamus, small crystals of the carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were applied to either graft or host after fixation of the brain. Tissue sections were analyzed for retrograde and and anterograde movement of the dye. When crystals were placed on the graft, labeled axons were found in the host median eminence or in the host hypothalamus taking an arching trajectory toward the median eminence. Retrogradely labeled neurons in the host were few in number and largely confined to the host arcuate nucleus. With DiI crystals applied to the basal hypothalamus, labeled axons were distributed widely in the host but much sparser in the graft. Axons appeared to enter primarily at sites where the graft and host interface lacked an ependymal lining. Small numbers of retrogradely labeled neurons were also seen in the graft. Most were cells of very simple morphology and were distributed randomly in the graft. When double label experiments were carried out most DiI positive cells in the graft contained GnRH. These results indicate the connectivity between host hypothalamus and the third ventricular preoptic area grafts exists but is limited in nature.