Studies on the Subsynaptosomal Localization of Luteinizing Hormone-Releasing Hormone and Thyrotropin-Releasing Hormone in the Rat Hypothalamus*

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Abstract
Subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH [luteinizing hormone releasing hormone], TRH [thyrotropin releasing hormone], and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal particles which banded at sucrose densities between 0.6-1.0 M. EM analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. NE was found in 2 pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with dense-cored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles.