ABSTRACT: Adult Neurogenesis and Gene Expression Changes in 5-HT7 Receptor Knockout Mice: In the adult, the formation of new nerve cells in the CNS is restricted to the subependymal layer and to the subgranular zone of the hippocampus (Duman et al., 2001). Clinically adult neurogenesis has received most attention for its possible role in major depression. Depressed patients have reduced hippocampal neurogenesis (Kasper & McEwen, 2008) and hippocampal volume (Colla et al., 2007). It has also been shown that chronic, but not acute, treatment with currently widely used antidepressants, most notably fluoxetine, results in increased hippocampal neurogenesis (Malberg et al., 2000; Miller et al., 2007). Pharmacologically antidepressants act by elevating the amount of synaptic serotonin (5-HT) and they do that within minutes of administration, but the clinical effect is often delayed, sometimes for weeks (Miller et al., 2007). This delay is believed to involve changes in plasticity and neurogenesis. With currently available treatment options for depression 20% or more of patients do not respond to the therapy. Thus, there is a need for an increased understanding of the mechanism behind plasticity and neurogenesis, and for the development of improved therapies for depression. We have in an ongoing project with the aim to functionally characterize the 5-HT7 receptor, one of the most recently discovered receptor subtypes for 5-HT (Lovenberg et al., 1993). Interestingly, we have found that this receptor might be a new target for the treatment of depression. Inactivation or pharmacological blockade of this receptor induces an antidepressant-like profile in several preclinical models of depression (Hedlund et al., 2005; Guscott et al., 2005; Wesolowska et al., 2006a, 2006b) and in sleep patterns (Thomas et al., 2003; Hedlund et al., 2005). Of special interest is also the finding that inhibition of the 5-HT7 receptor potentiates the effect of antidepressants in behavioral models of depression (Wesolowska et al., 2007; Bonaventure et al., 2007). We have recently determined that mice lacking lacking the 5-HT7 receptor show a 25% decrease in the number of proliferating cells in the subgranular zone of the hippocampus compared to wildtype mice. The mice were treated with BrdU, a thymidine analog that is incorporated into the DNA of cells undergoing mitosis. BrdU was then detected immunohistochemically. Both wildtype and knockout mice responded to fluoxetine treatment with an increase in the number of dividing cells. The reduction in the number of dividing cells in the 5-HT7 receptor knockout mice seemingly contradicts the antidepressant-like profile previously observed in these mice, but might on the other hand help to explain previously observed impairments in hippocampus-dependent learning seen in the knockout mice (Roberts et al., 2004) and after pharmacological blockade (Meneses, 2004). The observation that 5-HT7 receptors influence neurogenesis constitutes an entirely new regulatory mechanism for this physiologically and pathophysiologically important process. In order to further understand hippocampal neurogenesis and how it is influenced by 5-HT in general and 5-HT7 receptors in particular we propose to perform a microarray study using the glycogene array designed by the Consortium for Functional Glycomics using total RNA from the hippocampus. A comparison will be made between young adult male mice lacking the 5-HT7 receptor and wildtype sibling controls. Such a study should yield new valuable insights into the pathways regulating neurogenesis. The glycogene array is of particular interest as it includes several genes that have been shown to be either directly or indirectly involved in adult neurogenesis and to be regulated by 5-HT and antidepressants. Of particular interest are cell adhesion molecules which are glycoproteins. One such molecule is NCAM, a marker for immature neurons undergoing remodeling and plasticity (Nakagawa et al., 2002), which is upregulated by fluoxetine treatment (Varea et al., 2006). In contrast, NCAM is downregulated following 5-HT depletion (Brezun & Daszuta, 1999). Chronic fluoxetine treatment has also been shown to upregulate neuropeptide Y, Galectin I, Activin A receptor and Inhibin beta-A, all included in the glycogene array (Miller et al., 2007). Another relevant gene on the glycogene chip is ST8SiaIV (Nakagawa et al., 2002). Experimental Design: The hippocampus will be dissected and total RNA will be extracted according to recommended protocols for array studies. RNA from a sufficient number (2-3 animals) of male 5-HT7 receptor knockout mice will be pooled for each of the three replicates. Control samples will be similarly generated from wildtype siblings. The 5-HT7 receptor mice are on a genetically homogeneous background (backcrossed for 16 generation on a C57BL6/J background) so inter-animal variability should be negligible. The RNA will be analyzed on the glycogene array. 3 KO replicates + 3 WT replicates per glycogene array = 6 arrays
