Effect of NMDA-induced lesion of the subfornical organ on the angiotensin II binding sites density and acetylcholinesterase or NADPH-diphorase activities in the lamina terminalis of the rat brain.

Abstract

1. Neural angiotensinergic circuitry located in the lamina terminalis has been proposed to be involved in blood pressure regulation and fluid homeostasis. 2. ANG II binding sites have been described to be localized throughout the lamina terminalis including the subfornical organ (SFO), the median preoptic nucleus (MnPO), and the organum vasculosum lamina terminalis (OVLT). 3. The present experiment was designed to investigate the ANG II binding sites localization in the lamina terminalis. For this purpose, we have compared the ANG II binding sites, acetylcholinesterase, and NADPH-diaphorase distributions throughout the lamina terminalis. Additionally, we have studied the effect of the preferential lesion of SFO neuronal cell bodies by local injection of NMDA on the ANG II binding sites density in different areas of the lamina terminalis. 4. Male Wistar rats were anesthetized, immobilized in a stereotaxic apparatus, and 500 nl of saline or 250 nmol NMDA was injected into the SFO. 5. Animals were sacrificed 1 week later, the brain was removed, frozen, and sagittal 16 microm slices were cut in a cryostat. Alternate brain slices were incubated with [125I]-Sar1-ANG II for receptor autoradiography or histochemically stained for visualization of acetylcholinesterase and NADPH-diaphorase activities. Binding capacity was determined by computerized quantitative densitometry of autoradiograms. The intensity of histochemical reactions was measured as relative units obtained by computerized densitometry processing of the brain slices stained for either activity. 6. Acetylcholinesterase staining was mainly located in the SFO, with faint staining reaction in other areas of the lamina terminalis. NADPH-diaphorase staining was homogeneously distributed throughout the lamina terminalis. A significant positive correlation was observed between acetylcholinesterase and NADPH-diaphorase stainings in the SFO of control and NMDA-lesioned rats. 7. ANG II binding sites were localized throughout the lamina terminalis. A significant positive correlation was observed between the density of ANG II binding sites and the intensity of acetylcholinesterase or NADPH-diaphorase staining in the SFO of control and NMDA-lesioned rats. 8. The distribution of the NADPH-diaphorase staining was found to closely match the distribution of the ANG II binding sites in the lamina terminalis. 9. Neuronal lesion of the SFO caused significant reductions in the density of ANG II biding sites in the SFO (-68%) and the MnPO (-48%). No changes were observed either in the OVLT or outside the lamina terminalis in the superior colliculus. 10. The present results indicate the following: first, the presence of high levels of acetylcholinesterase staining in the SFO and of NADPH-diaphorase throughout the lamina terminalis; second, that ANG II binding sites in the SFO and possibly in the MnPO are localized in neuronal cell bodies; third, that SFO lesion did not affect the expression of ANG II binding sites in the OVLT, thus suggesting that these binding sites correspond to different angiotensinergic system: and finally, the existence of a striking correlation between the distribution of the ANG II binding sites and NADPH-diaphorase throughout the lamina terminalis, thus suggesting a interrelation between angiotensinergic and nitrergic systems in the lamina terminalis.