Regulation of neurogenesis in adult mouse hippocampus by cAMP and the cAMP response element-binding protein

Abstract

The cAMP cascade, including the cAMP response element-binding protein (CREB), is known to play an important role in neuronal survival and plasticity. Here the influence of this cascade on neurogenesis in adult hippocampus was determined. Activation of the cAMP cascade by administration of rolipram, an inhibitor of cAMP breakdown, increased the proliferation of newborn cells in adult mouse hippocampus. In addition, rolipram induction of cell proliferation resulted in mature granule cells that express neuronal-specific markers. Increased cell proliferation is accompanied by activation of CREB phosphorylation in dentate gyrus granule cells, suggesting a role for this transcription factor. This possibility is supported by studies demonstrating that cell proliferation is decreased in conditional transgenic mice that express a dominant negative mutant of CREB in hippocampus. The results suggest that the cAMP-CREB cascade could contribute to the actions of neurotransmitters and neurotrophic factors on adult neurogenesis.

Fig. 1.

Effect of rolipram administration on cell proliferation in the adult hippocampus (a–c). The number of BrdU-positive cells in the entire bilateral dentate gyri 24 hr (a, b), 2 hr (c), or 4 weeks (d) after BrdU administration. Repeated (14 d) rolipram administration increased the number of proliferating cells in the granule cell layer compared with vehicle-treated control mice (a, c). Acute (1 d) rolipram treatment had no effect on the number of newborn cells (b). Chronic rolipram increased the number of surviving cells in the granule cell layer relative to control mice 4 weeks after BrdU administration (d). The results are expressed as the mean ± SEM number of BrdU-positive cells in bilateral dentate gyri (n = 6 animals). *p < 0.05 compared with the corresponding control (Student's t test).

Fig. 2.

Effect of chronic rolipram administration on the morphology and distribution of proliferating cells in the adult hippocampus. As quantified in Figure 1, chronic rolipram treatment (b) increased the number of proliferating cells relative to control mice (a). BrdU-positive cells were mostly in the subgranular layer between GCL and hilus 24 hr after BrdU injection. c–e, Examples of proliferating cells 24 hr after BrdU administration. Nuclei of BrdU-positive cells were dark and irregular in shape. Many proliferating cells occurred in clusters (arrow in e). No apparent difference in morphology and distribution of proliferating cells was seen between rolipram-treated and control mice. Scale bars:a, b, 200 μm; c–e, 10 μm.

Fig. 3.

Effect of chronic rolipram administration on the morphology, distribution, and phenotype of surviving cells in the adult hippocampus. BrdU-positive cells 4 weeks after BrdU administration showed mature cell morphology within the GCL: sparse, multipunctate (a) or dark, uniform (b) BrdU-labeled nuclei with pale cytoplasm. Confocal images of triple-labeled cells with the mitotic marker BrdU (green; c, g), the glial marker S100β (red; d,h), the neuronal marker NeuN (blue;e, i), and merged images of the three labels (f, j) demonstrate cells with a neuronal (c–f) or glial (g–j) phenotype. Scale bars: a,b, 10 μm; c–j, 20 μm. Rolipram-treated and control mice did not differ in depth and morphology of BrdU-positive cells within the granule cell layer. The number of BrdU-labeled cells expressing either NeuN or S100β was determined for each group. The results are expressed as percentage and are the mean ± SEM of a total of 50 cells counted for each group. There was no significant difference between the control and chronic rolipram-treated mice.

Fig. 4.

a–d, Immunostaining of pCREB in the adult dentate gyrus of acute or chronic rolipram-treated mice. Confocal images with low magnification showed that pCREB-immunopositive cells were aligned in the deepest part of GCL in the control animals (a, c). Although the same immunostaining pattern was seen in the acute rolipram-treated mice (b), the entire granule cell layer was strongly stained with pCREB antibody in the chronic rolipram-treated mice (d). e–j, Double immunolabeling of BrdU (green) and pCREB (red) in the adult dentate gyrus of chronic rolipram-treated mice 24 hr after BrdU administration. Confocal images with high magnification showed that BrdU-labeled cells (arrows) did not show immunoreactivity of pCREB in either acute (e–g) or chronic (h–j) rolipram-treated animals. Scale bars:a–d, 200 μm; e–j, 20 μm.

Fig. 5.

Schematic diagram of the tetracycline-regulated gene expression system. Gene 1 encodes the tetracycline-regulated transactivator protein tTA under the control of CaMKIIα promoter. Gene 2 encodes the FLAG-tagged CREB mutant (mCREB) gene with modified phosphorylation site (Ser¹³³ to Ala) under the control of the tetracycline-responsive promoter TetOP. In the absence of doxycycline, a tetracycline analog, tTA binds to and activates TetOP and increases the expression of the downstream target gene mCREB. When doxycycline is added, it binds to tTA, causes a conformational change of tTA, and prevents it from activating TetOP, thereby turning off the expression of target genes. We used transgenic mice that received doxycycline in the drinking water during development (i.e., pregnant mothers received doxycylcine) to postnatal week 6, followed by 4 weeks without doxycycline.

Fig. 6.

Inducible expression of mCREB decreases cell proliferation in hippocampus. mCREB expression was detected in olfactory bulb (OB), cortex (Cx), piriform cortex (Pir), caudate putamen (CP), nucleus accumbens (Acb), amygdala (Amy), hippocampus (Hi), and cerebellum (Cb) but not in the thalamus (Th) and midbrain and medulla oblongata (a, b) in CaMKII-tTA × TetOP-mCREB bitransgenic mice. No immunoreactivity was shown in TetOP-mCREB single transgenic mice (c). In the dentate gyrus, most of, but not all, granule cells (d) and a few cells in (Figure legend continued.) the CA3 region expressed mCREB (d). Double immunolabeling for BrdU and mCREB in the adult hippocampus of bitransgenic mice 2 hr after BrdU administration is shown in f. There was no mCREB immunoreactivity in the BrdU-positive cells. Scale bars:a, 1 mm; b–d, 200 μm;e, f, 20 μm. Mice expressing mCREB in the hippocampus or genotype controls were treated with BrdU and 2 hr later harvested for immunohistochemical analysis. The number of proliferating cells in the granule cell layer was significantly decreased in the bitransgenic mice relative to single transgenic mice (g). CaMKII-tTA, CaMKII-tTA single transgenic mice with no mCREB expression; TetOP-mCREB, TetOP-mCREB single transgenic mice with no mCREB expression;CaMKII-tTA × TetOP-mCREB, CaMKII-tTA × TetOP-mCREB bitransgenic mice that express mCREB in hippocampus. *p < 0.05 compared with the single transgenic control (ANOVA, followed by Scheffe's post hoc comparison).