Simultaneous localization of LHRH and catecholamines in rat hypothalamus

The PAP unlabelled antibody enzyme method of Sternberger was used for the histochemical demonstration of LHRH and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) in the hypothalamus of the adult male and pregnant female rat. The sections for light and electron microscopy were serially treated with normal goat serum, LHRH antiserum and/or TH antiserum, goat anti-rabbit IgG, PAP complex and 3,3'-diaminobenzidine (DAB) or 4-Cl-1-naphtol. LHRH-positive cell bodies were discernible in the medial preoptic area. The LHRH-positive terminals were densely localized in the organum vasculosum of the lamina terminalis and in the perivascular region of the median eminence (PVME). Dopamine (DA)-positive cell groups (TH-positive perikarya) were discernible in the arcuate nucleus, and its terminals were densely localized in the PVME. The simultaneous identification of LHRH and DA in the distinctive neuronal system of the median eminence was possible with the PAP double staining technique, in which LHRH is revealed as a brown precipitate with DAB, and TH is revealed as a blue reaction product with naphtol. The LHRH neuronal system did not contain TH and vice versa. The ultrastructural study revealed that LHRH was localized in large vesicles with a diameter of 100 nm within the axon terminals, while TH was localized in the endoplasmic reticulum, the neurotubules and small vesicles with a diameter of 50 nm within the DA neuron. The axo-axonic contact of LHRH and DA terminals was demonstrated in close proximity to portal vessels, suggesting the synaptic influence of DA on the release of LHRH into these vessels.