Journal of Neurochemistry Lippincotl—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Differential Expression of Multiple Somatostatin Receptors in the Rat Cerebellum During Development ~ Viollet, C. Bodenant, C. Prunotto, tD. Roosterman, 1J. Schaefer, ~W. Meyerhof, *J. Epelbaum, H. Vaudry, and P. Leroux European Institute for Peptide Research, !FRMP 23, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, Mont-Saint-Aignan; *INSERM U159, Dynamique des Systèmes Neuroendocriniens, Paris, France; and t Department of Molecular Genetics, Deutsches Institut für Ernährungsforschungs, University of Potsdam, Potsdam, Germany Abstract: The present study describes the expression pattern of somatostatin receptor genes during the devel- opment of rat cerebellum. Characterization of somato- statin receptors was carried out by binding studies using receptor subtype-selective ligands in the germinative epi- thelium and granule cell layer of the cerebellum from post- natal day 4 (P4) to P21 and in granule cell cultures. Quan- titative reverse transcription-PCR carried out for the five receptor subtype mRNAs in cerebellar extracts showed that sstl mRNAs are predominant at the end of gestation. A transient high expression of the sst2 gene was ob- served from P7 to P14. In parallel, high levels of binding sites sensitive to sst2 ligands were detected in the gran- ule cell germinative epithelium and in granule cell cul- tures. sst3 mRNAs rapidly increased from P14 and be- came the predominant form at P21, but respective bind- ing sites were not detected. Whereas sst4 mRNA levels were generally low, those of sst5 were nearly undetect- able. Reverse transcription-PCR carried out in granule cell cultures revealed the relative abundance of sst mRNAs as follows: sst2 > sstl > sst3 = sst4. sst5 mRNA was undetectable. The results show the expression of four somatostatin receptor genes, but only three recep- tors (sstl, sst4, and mainly sst2) were detected as bind- ing sites during cerebellar development. Key Words: Reverse transcription-PCR—Germinative epithelium— Granule cells—Receptor binding—Cell culture. J. Neurochem. 68, 2263—2272 (1997). types (sstl—5) derived from five nonallelic genes lo- cated on separate chromosomes (for review, see Hoyer et al., 1995). Binding studies performed in tumor cells transfected with individual sst cDNAs have allowed the identification of somatostatin analogues selective for four receptor types (sstl, sst2, sst3, and sst5) (Raynor et al., 1993a,b; Patel and Srikant, 1994; Lia- pakis et al., 1996). In adult rats, somatostatin binding sites are distributed throughout the CNS in correspon- dence with SLI-containing areas (Epelbaum et al., 1985; Leroux et al., 1985; Reubi and Maurer, 1985). During development, high densities of somatostatin binding sites have been detected in immature areas, including the external granule cell layer of the cerebel- lum (EGC) (Gonzalez et al., 1988, 1989). These ob- servations indicate that somatostatin may exert activi- ties during brain development. Adult rat cerebellum contains virtually no somato- statin (Vincent et al., 1985; Epelbaum, 1986). In addi- tion, somatostatin receptors could hardly be detected in the cerebellar cortex by binding approaches (Piwko et al., 1995). Conversely, mRNAs coding for somato- statin (Inagaki et al., 1989; Bendotti et al., 1990; Naus, 1990) and SLI (Inagaki et al., 1982; Villar et al., 1989) are present in various cell types of the cerebellum dur- ing the first postnatal weeks. During this period, so- matostatin receptors are abundant in the EGC, the ger- Somatostatin is a tetradecapeptide widely distributed in the rat CNS. Somatostatin-like immunoreactivity (SU) is particularly abundant in the cerebral cortex, the limbic system, the hypothalamus, many brainstem nuclei, and the spinal cord (Johansson et al., 1984). In addition, SU is present for transient periods during the development of the CNS in areas that tend to re- gress or to undergo profound reorganization. Recent molecular cloning studies have demonstrated the existence of at least five somatostatin receptor sub- Received October21, 1996; revised manuscript received February 12, 1997; accepted February 13, 1997. Address correspondence and reprint requests to Dr. P. Leroux at European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U4l3, University of Rouen, 76821 Mont-Saint-Aignan Cedex, France. Abbreviations used: CH-275, desAA‘ 25[ D-Trp 8,lAmp9 I somato- statin-14; E, embryonic day; EGC, external granule cell layer of the cerebellum; GCL, granule cell layer; HEK293 cells, human embryonic kidney cells; 251-LDTT-S28, monoiodinated [Leu5,D- Trp22,Tyr25]somatostatin-28; P, postnatal day; RT, reverse transcrip- tion; SU, somatostatin-like immunoreactivity; SRIF, somatostatin; sst, somatostatin receptor subtype. 2263