G-CSF is specifically expressed by neurons in the rat CNS. (A–C) Double-immunofluorescence staining with the astrocytic marker GFAP revealed absence of G-CSF expression in astrocytes in the hilus of the dentate gyrus (A) or cortex (Cx; B). In contrast, there was perfect colocalization of G-CSF with cells expressing the neuronal marker NeuN (C). (D–I) In situ hybridization confirmed the neuronal expression of G-CSF and demonstrated an expression pattern that paralleled results obtained by immunohistochemistry. For example, G-CSF mRNA was detected in pyramidal neurons in the cortex (D; original magnification, ×40), in the hippocampus CA3 field (F; original magnification, ×40), and in specific cells located in or near the subgranular zone in the dentate gyrus (DG) (H; original magnification, ×40). Sense probes did not yield any specific staining in corresponding sections (E, G, and I; original magnification, ×40). (J) Using amplified mRNA from laser-excised neurons or astrocytes from the mouse cortex (100 cells each), a G-CSF–specific PCR signal could only be obtained in the neuronal pool but not from astrocytes after 50 amplification cycles. As a control, GFAP was amplified only from the astrocytic population, whereas the ubiquitous housekeeping gene cyclophilin was amplified from both cell pools. A brain cDNA library served as positive control (Pos.) for all PCR reactions. PCR reactions using water as input served as negative control (Neg.). M, size marker.