Figure 1.

CREB mRNA presence in growth cones and dendrites. (A) Representative reverse Northern blot probed with ³²P-CTP radiolabeled aRNA from a single dendritic growth cone. Slot a1, microtubule-associated protein 2; a2, CREB δ; a3, calmodulin-dependent kinase; b1, c-fos; b2, c-jun; b3, C82 (a C2H2 zinc finger cDNA); c1, zif268; c2, orthodenticle 1; c3, pBS. (B) Reverse Northern blot probed with ³²P-CTP radiolabeled aRNA from a single cell body. The arrangement of cDNAs is as described for A. (C) Localization of CREB mRNA by using digoxigenin-labeled CREB cRNA as a probe in cultured rat hippocampal cells by using in situ hybridization. The neuronal soma and the CREB positive processes exhibit a purple color because of NTB staining of the dig-labeled cRNA that is hybridized to CREB mRNA. A single CREB mRNA containing process is indicated by the arrow. The inset shows background staining when in situ hybridization is performed with no cRNA probe added to fixed cells. Bar = 15 μM. (D) Localization of CREB mRNA by using digoxigenin-labeled CREB cRNA as a probe in neurons from normal human temporal neocortex by using in situ hybridization. Apical dendrite of a layer V pyramidal cell (arrow). Several adjacent cell bodies contain abundant CREB mRNA within the soma (out of focal plane). Inset depicts competition control on an adjacent tissue section (arrow). Positive in situ hybridization in dendrites was observed in human brain neocortex from four individuals. The competition control shows the in situ hybridization signal produced when unlabeled antisense cRNA is added to prehybridization mix at a concentration of 50× the amount of labeled cRNA to be used in the hybridization step. Between prehybridization and hybridization the unhybridized unlabeled cRNA is washed away. Bar = 10 μM.