Figure 4

Assessment of NTPDase3-levels in the hypothalamus of female rats. A: Sample collection. Tissue blocks of the medial part of the hypothalamus (Med-HT) containing the arcuate nucleus and the lateral part of the hypothalamus (Lat-HT) containing the lateral hypothalamic nucleus were excised as follows: A coronal slice of the entire rat brain was cut with a rostral border at anterioposterior level 2.12 mm behind the bregma (just behind the caudal border of the optic chiasm) and a caudal border at anterioposterior level 4.52 mm behind the bregma (just before the caudal tip of the mamillary body). Slices were cut into two halves along the midsagittal plane. The dorsal border of the tissue block was cut along a horizontal line dorsally tangential to the third ventricle and the remaining cortical tissue and optic tract were removed. The remaining tissue block was further cut into two halves along a sagittal plane passing through the fornix. Thus, we obtained two tissue blocks from both the medial and lateral parts of the hypothalamus from each animal. B: Representative image showing NTPDase3-immunoreactive material in the Lat-HT and Med-HT four hours after a single subcutaneous injection of 17β-estradiol. Four immunoreactive bands were detected using homogenates of whole tissue samples. The 82–85 kDa molecular weight form of the enzyme, generally considered as the fully functional form, was further analyzed. Note the difference between the densities of the 82–85 kDa bands of the Lat-HT and Med-HT samples.