Cell Tissue Res (1990) 262:431-443 and Reseaw.h 9 Springer-Verlag 1990 Somatostatin-binding sites on rat teleneephalic astrocytes Light- and electron-microscopic studies in vitro and in vivo Rolf Mentlein, Cornelia Buchholz, and Brigitte Krisch Anatomisches Institut der Universit/it Kiel, Kiel, Federal Republic of Germany Accepted August 6, 1990 Summary. Using a somatostatin-gold conjugate as li- gand, high-affinity binding sites for this neuropeptide were demonstrated at three levels: (i) cultured astrocytes from rat cortex, (ii) hippocampal slice cultures, and (iii) frozen tissue sections of rat telencephalon. The conjugate proved as active as the native peptide in competing for the binding sites. Light-microscopic visualization of bound ligand was achieved by silver intensification of the colloidal gold. This method is faster and yields supe- rior resolution compared with autoradiography. Cul- tured astrocytes from cortex and hippocampus could be labeled by the ligand. At the light- and electron-mi- croscopic level, astrocytes could be double-labeled by the somatostatin-gold conjugate and immunostaining for glial fibrillary acidic protein (GFAP). In hippocam- pal slice cultures, the conjugate did not penetrate into the neuropil because of a covering glial layer. However, a portion of this completely GFAP-positive covering glia reacted with the somatostatin ligand. In frozen brain sections, apart from delicate punctate structures, two types of labeled glia cells were seen: single stellate astro- cytes and perivascular glia cells. Key words: Astrocytes - Telencephalon - Receptors, membrane - Somatostatin (SRIF) - Rat (Han: WIST) Apart from its role as a neuroendocrine release-inhibit- ing factor, the tetradecapeptide somatostatin (SRIF) is supposed to be a neurotransmitter or a neuromodulator. SRIF is widely distributed in the brain. It has been local- ized in synaptic transmitter vesicles (Krisch 1980), high- affinity binding sites have been detected biochemically in brain membranes (Srikant and Patel 1981) and dem- onstrated by autoradiography in sections of rat cortex (Pelletier et al. 1986). However, up to now, individual Send offprint requests to : Prof. Dr. Brigitte Krisch, Anatomisches Institut, Neue Universit/it, Olshansenstrasse40, W-2300 Kid, Fed- eral Republic of Germany target cells for SRIF (neurons or glia cells) have not been morphologically identified. This investigation utilizes a conjugate of SRIF to colloidal gold to visualize high-affinity binding sites on telencephalic astrocytes. The electron-dense conjugate has the advantage of showing binding sites at the cellular level, a resolution not obtained with radiolabeled li- gands. The conjugate of SRIF to colloidal gold has re- cently been shown to be biologically active in modulat- ing the release of growth hormone from cultured adeno- hypophyseal cells and useful in visualizing high-affinity binding sites on these cells at the light- and electron- microscopic level (Mentlein et al. 1989). Telencephalic astrocytes are possible target cells for SRIF. Calker et al. (1980), and Cholewinski and Wilkin (1988) have reported that SRIF modulates the agonist- induced elevation of intracellular cyclic AMP in cultured astrocytes from rat cerebral cortex. Therefore, we evalu- ated the occurrence of high-affinity binding sites for SRIF on three levels, namely, cultured astrocytes from cerebral cortex and from hippocampus, organotypic hip- pocampal slice cultures, and sections of rat telen- cephalon. Materials and methods Materials Somatostatin-14 was purchased from Bachem (Bubendorf, Switzer- land), bacitracin from Serva (Heidelberg, FRG), aprotinin (Trasy- lol) from Bayer (Leverkusen, FRG), soybean trypsin inhibitor and phosphoramidon from Sigma (Miinchen, FRG), Sephadex S-300 gel was obtained from Pharmacia (Freiburg, FRG). SMS 201-995 was a gift from Sandoz (Basel, Switzerland). Gey's balanced salt solution was purchased from Gibco (Eggenstein, FRG), other cell culture chemicalswere from Gibco and Nunc (Wiesbaden, FRG). Preparation of the SRIF-gold conjugate The SRIF-gold conjugate was prepared by a modification of the procedure described earlier (Mentlein et al. 1989). Colloidal gold