In situ hybridization: Male and female CD-1 mice, 3-4 week old were used. Clones were purchased from Research Genetics when available, or templates for probes were synthesized by PCR using specific primers and cDNA from mouse brain. For some genes, sense probes were also synthesized to control for non-specific hybridization. Digoxigenin- labeled RNA probes were made and hybridization was performed essentially as previously described (1), with some modifications. Briefly, fresh frozen, 20 mm thick coronal sections were cut with a cryostat.Ý Sections were dried and fixed in 4% paraformaldehyde, washed in PBS and subjected to acetylation using 0.25 % acetic anhydride in 1M Triethanolamine-HCl pH 8.0.Ý Slides were prehybridized for 1- 3 hr, and hybridized overnight at 70ƒC, using a probe concentration of 0.5 - 1 µg/ml.Ý Sections were washed twice in 0.2X SSC at 70ƒC for 30 min., incubated with anti-digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche) at a 1: 2000 dilution in 0.1M maleic acid buffer, pH 7.5, with 0.2 % Tween-20, 20 % sheep serum and 2 % blocking reagent (Roche). Staining was developed for 4 - 16 hours with NBT and BCIP (Roche) in alkaline phosphatase bufferÝ to yield a purple product.Ý Slides were fixed in 4% formaldehyde and mounted with glycerol.Ý To aid in the visualization of brain regions, Nissl staining was done on adjacent sections.

1.ÝÝÝÝÝÝÝÝ Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. & Ish-Horowicz, D. (1995) Nature 375, 787-790.